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HUE UNIVERSITY
UNIVERSITY OF SCIENCE
DANG THI THANH HA
THESIS OF DOCTOR OF BIOTECHNOLOGY
RESEARCH TO CREATE REcombinant LACCASE

AND TEST FOR COLORS
SOME INDUSTRIAL Dyestuffs
Hue, in 2022
HUE UNIVERSITY
UNIVERSITY OF SCIENCE
DANG THI THANH HA
RESEARCH FOR CREATION OF REcombinant LACCASE AND TESTING

The ability to decolorize some INDUSTRIAL Dyestuffs
Industry: Biotechnology
Code: 9420201
THESIS OF DOCTOR OF BIOTECHNOLOGY
Science instructor
1. Assoc. Dr. Pham Thi Ngoc

Lan 2. Dr. Nguyen Duc Huy
Hue, in 2022
TABLE OF CONTENTS
AUTHORITY ................................................................................. ................................. .... iv
THANK YOU ............................................... ................................. ....v
LIST OF ACRONYMS............................................. ..................................... en LIST OF
TABLES. ................................. .............................. ix LIST OF
PICTURES ...... ................................. ................................................x
INTRODUCTION... ................................. ................................. ..............first
1. URGENTITY OF THE TOPIC .................................................................. ................................first
2. RESEARCH OBJECTIVES................................................................................. ............................2 3.
RESEARCH CONTENT... ................................. ................................2 4. NEW CONTRIBUTIONS OF THE
THESIS ....... .................................3 Chapter 1. TOTAL DOCUMENTARY
REVIEW ............................................................................................... ............4
1.1. LACCASE ....................................................................................... ................................. .........4
1.1.1. General introduction to laccases ..................................................................................... .................................4
1.1.2. Structure of the laccase .......................................................................... .................................4 1.1.3.
Catalytic mechanism of laccases .............................................................................. .................................6
1.1.4. Characterization of laccases ....................................................................................... ............................................8
1.1.5. Sources of laccase receipts ..................................................................................... ............11 1.1.6.
Effect of culture conditions on laccase biosynthesis .......14 1.1.7. Applications of
laccases ....................................................................................... ................................................15 1.2. Pichia
pastoris EXPRESSION SYSTEM ............................................................................ ............18 1.2.1. General
introduction to the P. pastoris system ..................................................................... ...........18 1.2.2. Advantages
and disadvantages of P. pastoris compared to other expression systems .......19 1.2.3. Homologous
recombinant expression system using P. pastoris .......................20 1.3. INDUSTRIAL DYNAMICS AND
TREATMENT MEASURES .......25 1.3.1. Industrial dyes ....................................................................................... ............25
1.3.2. Methods of treating industrial dyes ................................................................27 1.3.3. Color treatment
of dyes with laccase .................................................................................. .................28 Chapter 2. RESEARCH
SUBJECTS AND METHODS .................................................................. ......33
i
2.1. RESEARCH SUBJECTS .............................................. ............33 2.1.1.
Object ................................................ ................................. ......33 2.1.2. Sample
collection ................................................................................................. ................................. ..33 2.2.
RESEARCH METHODS.............................................. ............33
2.2.1. Isolation of fungal strains with laccase activity .33 2.2.2. Investigation of laccase biosynthesis from
isolates .......................................34
2.2.3. Determination of laccase enzyme activity ....................................................................... ............35
2.2.4. Methods for the determination of extracellular proteins. ............35 2.2.5. Determination of some
characteristics of crude laccase from isolates ............36 2.2.6. Molecular identification of isolated fungal
strains ....................................................... ............36
2.2.7. Isolation of laccase-coding genes ............38 2.2.8. cFolac1 expression in Pichia
pastoris ..................................................................... ............40 2.2.9. Investigating factors affecting the
expression of recombinant laccase ..........43 2.2.10. Purification of the recombinant
enzyme ....................................................................... ............44
2.2.11. SDS-PAGE electrophoresis .................................................................. .............45
2.2.12. Investigate some factors affecting recombinant laccase activity ............45 2.2.13. Test for the
ability of synthetic dyes to decolorize .......................................46 2.2.14. Statistical
processing ....................................................................................... ............................47 Chapter 3.
RESEARCH RESULTS ............................................................................ ............48 3.1. Isolation of
LACCASE ACTIVE MOLD STRATEGIES ............48 3.1.1. Isolation, purification and breeding of mold
strains .......................................48 3.1. 2. Screening for laccase biosynthetic mold
strains ....................................................... 49 3.1 .3. Some morphological characteristics of colonies and
spore-forming stalks of fungal strains
isolation ................................................................................. ................................. .......50 3.1.4. Evaluation
of laccase-producing ability of isolated fungal strains .......................51 3.1.5. Investigating some
properties of natural laccases ..................................................................................... ........58
3.1.6. Identification of microorganisms ....................................................................................... .............60
3.2. CREATION OF LACCASE ENGLISHING GENES FROM F. oxysporum ..63 3.2.1. Amplification of
laccase genes from the cDNA of F. oxysporum ................................................63
ii
3.2.2. Cloning of genes encoding the enzyme laccase in the vector pGEM®T-Easy........64
3.2.3. Sequencing the laccase
gene ....................................................................................... .................................71 3.3.
Expression of the LACCASE ENCORDING GENES IN Pichia pastoris..77 3.3.1. Cloning
cFolac1 into pGEM®T-Easy and transforming into E. coli Top10.........77 3.3.2. Cloning into the
expression vector pPICZÿA ................................................................................ ............77 3.3.3. Optimizatio
3.3.4. Enzyme
purification ....................................................................................... ................................................86
3.3.5. Characterization of recombinant enzymes ....................................................................... ................87
3.4. TESTING ABOUT RECOMMENDED LACCASE INDUSTRIAL
Dyestuffs .................................................................................. .................................................
91 Chapter 4. WOMEN
DISCUSSION ................................................................................. .................................95 4.1.
Isolation of LACCASE ACTIVE MOLD STRATEGIES ............95 4.1.1. Culture
medium.............................................. .......................................95 4.1.2. Investigating some
properties of natural laccases ..................................................................................... ........96 4.2. Isolation of
pastoris ................................................................................. ................................. .................100
4.3.1. Folac1 gene
expression ............................................................................................... .................................100
4.3.2. Characterization of recombinant enzymes ....................................................................... .......102
4.3.3. Optimization of recombinant laccase expression. ............103 4.4. TESTING ABOUT
RECOMMENDED LACCASE INDUSTRIAL
Dyestuffs .................................................................................. .................................................106
CONCLUSIONS AND RECOMMENDATIONS. ................................. ................................................109
1. CONCLUSION.......... ................................. .................................................109 2.
RECOMMENDATIONS ..... ................................. ............................109 DISCLAIMER RELATED
TO THE THESIS .......................110 REFERENCES ................................. ............111
APPENDIX................................................ ................................. …………126
iii
GUARANTEE
I hereby declare that this is my own research work. The data and
results stated in the thesis are honest, objective, serious and have never
been published in any other works. If there's something wrong, I apologize
full responsibility.
Thesis author
Dang Thi Thanh Ha
iv
THANK YOU
First of all, I would like to express my sincere and deep gratitude to Assoc. Prof. Dr. Pham
Thi Ngoc Lan and Prof. Dr. Nguyen Hoang Loc, Department of Biology, University of Science, Hue
University for guiding and guiding me throughout the research process.
I would also like to express my sincere and deep gratitude to Dr. Nguyen
Duc Huy, Head of Enzyme and Protein Technology Laboratory, Deputy Director
Institute of Biotechnology, Hue University is a teacher who directly guides and supports business activities
expenses during the implementation and completion of this thesis.
Sincere thanks to the Board of Directors, Graduate School, Department of Biology,
University of Science, Hue University.
Thank you to the staff and students of the Laboratory
Biotechnology, Department of Biotechnology, Department of Applied Biology, have enthusiastically
guided and helped me throughout the study and research process in the department.
I would like to thank the staff and students of the Enzyme and Protein Technology
Laboratory, Institute of Biotechnology, Hue University for their enthusiastic guidance and creating
favorable conditions throughout the research process. at the Institute.
In addition, I would also like to thank Mr. Seung-Moon Park and MSc. Nguyen Thi My Le,
Chonbuk National University, Korea has provided some support
analysis during the completion of the thesis.
Finally, I would like to thank the Board of Directors of Tay Nguyen University, along with
family, friends and colleagues for encouraging, encouraging and creating the best conditions.
let me study and research.
Thank you very much!
Thua Thien Hue, ……month……..year 2022
Postgraduate
Dang Thi Thanh Ha
v
LIST OF ACRONYMS
2,4- EASY 2,4-Dichlorophenoxyacetic acid
2,4,5-BILLION 2,4,5- trichlorophenoxyacetic acid
ABTS 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)
Amp Ampicillin
BPA Bisphenol A
Bp Base pair
BSM Bacteria Standard Medium
CHCl3 Chloroform
COD Chemical Oxygen Demand
CTAB Cetyltrimethyl ammonium bromide
DMP 2,6-dimethoxyphenol
DNA Deoxyribonucleic acid
DW Distilled water (Sterile distilled water)
EC Enzyme commission
EDTA Ethylene diamine tetra-acetic acid
EMS (Ethyl Methane Sulfonate)
EP Eppendorf
EtOH Ethanol
Folac Laccase gene
HBT Hydroxybenzotriazole
Because
HPI N-hydroxyphtaimide
ITS Internal transcribed spacer
IUB International Union of Biochemistry
LB
Luria Bertani
LipP
Lignin peroxidase (ligninase)

MnP
Manganese peroxidase
NCBI
National Center for Biotechnology Information (China)
heart
DO
US National Biotechnology Information)
PAH
Dissolved Oxygen (Dissolved Oxygen)
PCB
Polycyclic aromatic hydrocarbon
PCI
Polychlorinated biphenyl
PCR
Phenol: chloroform: isoamylalcohol
PDA
Polymerase chain reaction
PVA
Potato Dextrose Agar
RACE PCR
Poly vinyl alcohol
RBBR
Rapid Amplification of ADNc-PCR
SDS – PAGE
Remazol Brilliant Blue CHEAP
TAE
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Taq
Tris-acetate-EDTA
TNT
Thermus aquaticus
vii
UV 2,4,6-trinitrotoluene
YP Ultraviolet (Ultraviolet Rays)
YPD Yeast peptone
YPDS Yeast peptone dextrose
YPM Yeast peptone dextrose sorbitol
Yeast peptone methanol
viii
LIST OF TABLES
Table 1.1. Application of laccase in dye biodegradation ..................................30 Table

2.1. Nucleotide sequences of primers used to amplify genes Folac1, Folac2,....39 Table
2.2. Sequences of primers used to amplify the cFolac1 gene from cDNA....41 Table 2.3.
Sequences of primers used to amplify AOX1F and AOX1R genes ................................42
Table 3.1. Protein content and laccase activity of 3 strains F4, F5, F8 ............51 Table
3.2. Accumulation of laccase of strain F4 in 4 culture media ............58 Table 3.3.
Decoloration rate of synthetic dyes of recombinant laccase .......................................91

Table 3.4. The role of mediators on the dye color processing ability of recombinant
laccases. ................................. .......................93
ix
LIST OF PICTURES
Figure 1.1. Spatial structure of laccases from Melanocarpus albomyces. .................5
Figure 1.2. Modeled laccase active site ............................5
by Sergio [10]. ................................. ................................. .......5 Figure 1.3. Structure of the active site
of laccase [60]. .................................6 Figure 1.4. Types of laccase catalysis ............7
Figure 1.5. Oxidation of ABTS to ABTS+ form under the catalysis....9
of laccases [88]. ................................. ................................. ......9 Figure 1.6. The pPICZÿ expression
vector system A, B, C for P. pastoris.....................................21
(Invitrogen, USA) [123]. ................................. ............................21 Figure 1.7. Mechanism of enzymatic
decolorization and degradation of azo dyes [79]. .......28 Figure 3.1. Colonies of 12 mold strains isolated
from Thua Thien Hue province
(F1-F12) ................................................................................. ................................. ................................48
Figure 3.2. Screening for peroxidase biosynthetic strains on supplemented selective media
guaiacol figs ..................................................................................................... ................................. ...........49
Figure 3.3. Mycelial colonies of strains F4, F5, F8 ............................................................ …………50
Figure 3.4. The ability to accumulate laccase of strain F5 in 4 fermentation media ...........52
Figure 3.5. The ability to accumulate laccase of strain F8 in 4 fermentation media ...........53 Figure 3.6.
The ability to accumulate laccase of strain F4 using MF3 . culture medium
at different temperatures ....................................................................................... .............54
Figure 3.7. The ability to accumulate laccase of strain F4 using MF4 . culture medium
Figure 3.10. The ability to accumulate laccase of strain F8 at 3 temperatures in the environment
MF3 fermentation ....................................................................................... ................................. ............56
x
Figure 3.11. The ability to accumulate laccase of strain F8 at 3 temperatures in the environment
MF4 fermentation ....................................................................................... ................................. ............57
Figure 3.12. Effect of pH on the laccase activity of strain F4 ................................................59
Figure 3.13. Effect of temperature on the laccase activity of strain F4 ..................59 Figure
3.14. Effect of metal ions on F4 laccase activity.................................................60 Figure 3.15.
Electrophoresis of total DNA separation product of F4.................................................61 Figure
3.16. PCR product ITS sequence 1-4 strain F4. ..................................62 Figure 3.17. Species
phylogenetic tree of strain F. oxysporum HUIB02 and some

other Fusarium species on Genbank. ................................. ............63
Figure 3.18. Electrophoresis of total RNA products from F. oxysporum HUIB02 ..........63 Figure
3.19. PCR products amplifying laccase-encoding genes.................................................64 Figure
3.20. PCR product of Folac1 gene from recombinant colonies.................................................65
Figure 3.21. PCR product of Folac2 gene from recombinant colonies.................................................65
Figure 3.22. Folac3 gene PCR products from recombinant
colonies ................................................................ 66 Figure 3.23. Folac4 gene PCR product from
recombinant colonies.................................................................66 Figure 3.24. Restriction product by EcoRI reco

Folac1................................................................................. ................................. ............68
Figure 3.25. Restriction product by EcoRI recombinant plasmid pGEM®T-Easy

Folac2................................................................................. ................................. ............69

Folac3................................. ................................. ............70
Figure 3.27. Restriction product by EcoRI recombinant plasmid pGEM®T- Easy

Folac4................................................................................. ................................. ............71
Figure 3.28. Genealogical tree of Folac1 gene and some other laccase genes ............72
on Genbank. ................................. ................................. ........72
Figure 3.29. 3D spatial structure model of Folac1 F. oxysporum HUIB02.

................................. ................................. ............72
Figure 3.30. Genetic genealogical tree of Folac2 gene and some other laccase genes ............73
on Genbank. ................................. ................................. ........seventy three
xi

................................. ................................. ............74
Figure 3.32. Genealogical tree of Folac3 gene and some other laccase genes ............75
on Genbank. ................................. ................................. ........75
Figure 3.33. 3D spatial structure model of Folac3 F. oxysporum HUIB02

................................. ................................. ............75
Figure 3.34. Genetic genealogical tree of Folac4 gene and some other laccase genes ............76
on Genbank. ................................. ................................. ........76

................................. ................................. ............77
Figure 3.36. EcoRI and XbaI cleavage products .....................................................................

78 Figure 3.37. Purified product of the cleavage reaction by EcoRI and
XbaI ............................78 Figure 3.38. PCR product cFolac1 using primer pair
AOX1. .............79 Figure 3.39. Recombinant Folac1 activity over induction
time. ............80 Figure 3.40. Recombinant Folac1 activity according to cell
density. .................................81 Figure 3.41. Recombinant Folac1 activity according
to the concentration of methanol inducer ............82 Figure 3.42. Recombinant Folac1
activity according to culture temperature. .................................84 Figure 3.43.
Recombinant Folac1 activity according to shaking speed. 85 Figure 3.44.
Electrophoresis of the recombinant protein product of rFolac1. ............87 Figure 3.45.
Effect of pH on the stability of recombinant laccase. ..........................88 Figure 3.46.
Effect of temperature on the stability of recombinant laccase .......................................88
Figure 3.47. Effect of metal ions on the stability of the recombinant laccase ......90 Figure 3.49. De
xii
PREAMBLE
1. URGENTITY OF THE TOPIC
Biotechnology has been changing the world, so many organizations and experts
around the world have affirmed that the 21st century is the century of Biotechnology.
Along with the development of society, in recent years, the field of biotechnology has
increasingly affirmed its role. effective in many fields such as industry, agriculture,
medicine - pharmacy, materials... with the creation of new varieties of plants and animals
for productivity, quality with high economic efficiency, enzymes created produce biological
products for the treatment of diseases, and microbial products
applications in environmental remediation.
With the rate of environmental pollution increasing due to the uncontrolled discharge
of wastes into the environment today, it is increasingly difficult to use conventional

chemical and biological methods to achieve the necessary levels to remove them. Pollutants.
Therefore, it is imperative to develop effective treatment methods that do not cause
secondary pollution. Recent studies have demonstrated that enzymes have many
possibilities and prospects in solving the problem of environmental pollution.

Enzymes can act on pollutants that are particularly difficult to handle to remove them by
precipitation, conversion, and decomposition of pollutants into other forms. In addition,
enzymes can change the properties of wastes to make them easier to handle or convert
into more valuable products. The enzyme treatment method has the following advantages:
applied to difficult biological substances, effective even in areas of high concentration of
environmental pollutants, a number of specific enzymes have a wide range of effects.
pH, temperature, ... without causing abnormal changes, without causing obstacles to
disrupt the ecological balance. As is known, toxic substances in the environment are
usually aromatic organic substances such as phenol compounds, cyclic amines or
phosphorus compounds, which in order to achieve the purpose of environmental

treatment must be destroyed or eliminated. those toxins. Among enzymes, the enzyme that reacts w
first
class 1 redox (oxydoreductase) and class 3 hydrolytic enzymes (hydrolase) have very high
ability to degrade the compounds mentioned above.
Laccase (EC 1.10.3.2, p-diphenol oxidase) is a particularly common and versatile
enzyme widely and diversely produced in nature from plants, fungi, bacteria and insects,
belonging to the oxidase family of enzymes. , namely phenol oxidase, catalyzes the
oxidation of many organic compounds including diphenols, polyphenols, diamines, aromatic
amines, benzenethiols and some inorganic compounds such as iodine, etc. Laccase has
strong oxidizing properties and has a broad spectrum. With diverse substrates and using
molecular oxygen as an electron acceptor, this enzyme is widely used in industry, in the
treatment of industrial - agricultural by-products and polluted wastewater. Besides, laccase
is also an environmentally friendly enzyme because in the laccase reaction, it only needs
to take oxygen from the air and the only byproduct formed after the reaction is water.
With such important applications and advantages, we chose the topic: "Research on
creating recombinant laccases and testing the ability to decolorize some industrial
dyes" in order to create recombinant laccases. combination to decompose organic
compounds that are difficult to decompose causing environmental pollution.
2. RESEARCH OBJECTIVES
Theoretical
objectives - To select new strains of microorganisms capable of strong laccase resolution.
- Study on laccase expression of Fusarium oxysporum in Pichia pastoris
to improve biosynthetic efficiency.
- Successful degradation of some synthetic dyes by recombinant laccase.
Experimental objective: to create recombinant laccase to degrade persistent organic
compounds causing environmental pollution.
3. RESEARCH CONTENTS -
Selection of laccase biosynthetic fungi; - Generating
laccase gene into the expression vector system for P. pastoris; -
Biosynthesis of laccase in P. pastoris; -
Purification and characterization of the recombinant laccase;
2
- Investigate the ability of recombinant laccase to decompose organic color compounds (difficult to
decompose).
4. NEW CONTRIBUTIONS OF THE THESIS - Isolated strain
F. oxysporum HUIB02 with strong ability to biosynthesize extracellular laccase. This is the first study
in Vietnam on laccase from F. oxysporum.
- Successfully cloned and sequenced 04 genes encoding laccase from DNA and cDNA of strain F.
oxysporum HUIB02. The genes with high similarity to the laccase gene group of F. oxysporum strains
isolated from other regions of the world showed a high conservatism in the laccase gene group in fungi in
general and F. oxysporum said.
private.
- Successful recombinant expression of the gene encoding laccase from the cDNA of strain F.
oxysporum HUIB02 in P. pastoris. The gene is denoted with the symbol Folac1, which is the first study in
the world on successful recombinant expression of Folac1 in the host.
- Natural laccase and recombinant laccase are capable of degrading the color of many synthetic
dyes with an efficiency of more than 90%. The results demonstrate the high applicability of recombinant
laccase in the treatment of organic compounds causing environmental pollution.
3
Chapter 1. DOCUMENTARY OVERVIEW
1.1. LACCASE
1.1.1. General introduction about laccase
Laccase (Lacc, EC 1.10.3.2) belongs to the group of polyphenol oxidase, is a protein
containing copper (Cu) in structure, with the characteristic features of oxidizing aromatic compounds
and requiring molecular oxygen for activity. Laccase is also known as an environmentally friendly
enzyme because in the laccase reaction it only needs to take oxygen from the air and the only
byproduct formed after the reaction is water [80].
Laccase is one of the few enzymes that has been studied since the nineteenth century.
Yoshida first described laccase in 1883 when he extracted it from the secretions of
Japanese lacquer tree (Rhus vernicifera). In 1893, Gabriel Bertrand isolated
obtained this enzyme from R. succedanea and other strains of the Anacardiaceae family:
Mangifera indica, Schinus molle, Pistacia palaestina, Pleiogynium timoriense.
Laccase is an enzyme of the Cu nuclear protein group, several other enzymes in the Cu nucleus
include cytochrome oxidase in plants and ceruloplasmin in mammalian plasma. Types of laccases
extracted from different sources have different levels of glycosylation, molecular weight and enzyme
kinetics [43].
1.1.2. Structure of laccase
1.1.2.1. Molecular mass Laccase
molecules are usually monomeric proteins, only a few are oligomeric proteins, with molecular
weights ranging from 60 to 90 kDa. Most of the fungal laccases are glycoproteins with a
carbohydrate content of about 10-25% [24].
1.1.2.2. Space structure
Laccase is classified as a Cu nuclear protein with four Cu atoms in three different redox
states. The laccase molecule normally consists of three main subunits (regions) A, B, and C with
relatively equal mass, all three of which play a role in laccase catalysis. The substrate binding site
is located in the cleft between the B . region
4
and C, the one Cu center is located in the C region and the three Cu center is located at the
common surface of the A and C regions (Figure 1.1) [13].
Figure 1.1. Spatial structure of laccases from Melanocarpus albomyces.
Subsections A, B, and C are designated red, yellow and green [13].
As shown in Figure 1.1, the single-atom Cu center contains only one Cu T1 atom, bound
to a peptide fragment with 2 histidine and 1 cysteine residues. The bond between the T1
copper atom and the S atom of cysteine is a stable covalent bond and absorbs light at 600 nm,
giving the laccase its characteristic blue color.
Figure 1.2. Modeled laccase active center
by Sergio [10].
5
All laccases are similar in structure to the catalytic center with 4 Cu atoms.
These Cu atoms are divided into 3 groups: type 1 (T1), type 2 (T2) and type 3 (T3), which differ in their
light absorption and electron potential properties. Cu atoms T1 and T2 have electron adsorption
properties and form a strong electron spectrum, while the Cu T3 atom pair does not form an electron
absorption spectrum and can be activated when bound to a strong anion [43] ], [60], [80].
Figure 1.3. Structure of the active site of laccase [60].
The Cu center has 3 atoms including a Cu T2 atom and a pair of Cu T3 atoms. The Cu T2 atom
binds to 2 conservative histidine moieties while the Cu T3 atom binds to 6 conservative histidine
moieties [60].
1.1.3. The catalytic mechanism of laccase
Laccase is a redox enzyme capable of oxidizing diphenols and related compounds, using
molecular oxygen as an electron acceptor. The redox potential of laccase ranges from 0.4 V to 0.8 V
[132]. A reducing substrate that loses an electron by the laccase catalysis usually forms a free radical,
which is further oxidized by the laccase catalysis or continues the reaction without
enzymatic catalysis such as hydration, dissociation or polymerization [10].
The single-atom Cu center (T1) is where the oxidation of the substrate takes place. The substrate
transfers an electron to the Cu T1 atom, turning the Cu T1 atom (Cu2+) into
6
Cu+ form , forming a laccase molecule with all 4 Cu atoms in the reduced state (Cu+ ).
A catalytic cycle involves the simultaneous transport of 4 electrons from a Cu T1 atom to
a cluster of Cu T2/T3 atoms across a conserved tripeptide bridge His-Cys-His. The
oxygen molecule then oxidizes the reduced laccase, forming a peroxidase intermediate
that is eventually reduced to water [10], [43].
Alternatively, laccase catalysis can occur by the following mechanism. First, the
substrate is oxidized directly by the active center of 4 Cu atoms. However, the substrate
molecules are often bulky or have too great a reduction potential, so they cannot reach
the reaction center of the laccase molecule. In this case an intermediate chemical
compound is needed. This chemical compound can come into contact with the reaction
center of the laccase and be oxidized by the laccase to the free radical form. Then the
chemical intermediate in the oxidized form accepts an electron from the substrate and
becomes the reduced form, continuing to participate in the catalytic cycle. In contrast, the
laccase, after giving the intermediate chemical compound an electron, becomes the
reduced form and then oxidizes to the oxidized form and continues to participate in the next catalytic
1.4).
Figure 1.4. Types of laccase catalysis [10].
(a): Catalytic cycle without the participation of chemical intermediates. (b); (c):
Cyclic catalysis with the participation of chemical intermediates.
The chemical intermediates commonly suitable for laccases are 3-
hydroxyanthranilic acid (HAA), 2,2'-azino-bis 3-ethylbenzthiazoline-6-sulphonic
acid (ABTS), N-hydroxybenzo-trialzone (HBT), N-hydroxyphtaimide (HPI), violuri
7
acid (VLA)… The participation of chemical intermediates has increased the catalyst substrate spectrum
and the substrate nonspecificity of laccase [10].
1.1.4. Characteristics of laccase
1.1.4.1. Inhibitors of laccase . activity
Inhibitors of laccase are usually small ions such as azide, cyanide, fluoride.
These ions will bind to the center of Cu 3 atoms and hinder the flow of electrons to these atoms. Other
laccase inhibitors are ethylene diamine tetra-acetic
acid (EDTA), fatty acid, tropolone, kojic acid and coumaric acid, etc., but they only have inhibitory effect
at high concentrations. Sulfhydryl-containing compounds such as L-cysteine, dithiothreitol and
thioglycolic acid are also considered laccase inhibitors. In addition, there are a number of other inhibitors
such as: some metals, L-cysteine, glutathione, dithiothreitol and thiourea. More recent studies have
shown that inhibition of laccase by metal shows binding of chloride and fluoride anions to the Cu T2
center and thereby inhibits oxygen reduction [133].
1.1.4.2. Mechanistic specificity
The substrate specificity of laccase is low because laccase has a very broad substrate spectrum.
Laccase has ortho and para-diphenol activity while tyrosinase has only o . activity
diphenols. Therefore, only tyrosinase has cresolase activity (oxidizes L-tyrosine).
and only laccase was able to oxidize syringaldazine. Lacacse is capable of oxidizing 2,2'-azinobis-bis-(3-
ethylbenzthiazolinesulphonate) (ABTS) to the strong light-absorbing ABTS+ cation state at 420 nm. The
higher the cation concentration, the
The darker the blue color, the stronger the enzyme activity (Figure 1.5). In addition, low substrate
specificity is also reflected in the wide range of laccase substrates. Hydroquinone, catechol, guaiacol
and 2,6-dimethoxyphenol (DMP) are all good substrates for laccase [134].
Laccase can oxidize both methoxy polyphenols and many other compounds. The suitability of
substrates for laccase is determined by two main factors. The first is the compatibility between the
substrate and the Cu T1 atom, the second is the dependence on the difference between the redox
potential between the substrate and the enzyme. These quantities depend on the chemical structure of
the substrate [4].
8
Figure 1.5. Oxidation of ABTS to ABTS+ under the catalysis
of laccases [88].
1.1.4.3. Effect of temperature and pH
The temperature stability of laccases varies considerably, depending on the source of
the microorganism. In general, laccases are stable at 30°C – 50°C and rapidly lose their activity
at temperatures above 60°C. Laccases from different strains will have different optimal
temperatures. According to some studies, 25ºC is the optimal temperature for laccase production
under light conditions and in the absence of light, the optimal temperature is 30ºC. The optimum
temperature range for laccase production is between 25ºC and 30ºC. Farnet et al (2008)
reported that, when incubating enzymes at 40°C and 50°C, laccase activity increased greatly. Laccase from
Pichia ostreatus was highly active in the temperature range of 40ºC - 60ºC, with the greatest
activity at 50ºC and remained unchanged after prolonged incubation at 40ºC for 4 h [26].
Effect of pH: Optimal pH values vary depending on the substrate that reacts with the
laccase. Laccase is optimally active in the pH 4.0 – 6.0 range for phenolic substrates. When
increasing the pH to the neutral or alkaline region, the activity of laccase is reduced, caused by
the small anion, which is the hydroxide ion (OH¯ ) bound to the Cu T1/T2 centers, inhibiting the
laccase. On the other hand, increasing pH also reduces the redox potential of phenolic
substrates so that phenolic substrates are more easily oxidized by laccase. Laccase activity at
different pHs is the result of two opposing effects,
9
one side is an increase in the laccase-substrate redox potential difference and the other side
is an inhibitory effect on the center of the Cu atom of hydroxide ion (OH¯) [69 ], [126].
When using the syringaldazine substrate and determining the effect of pH on enzyme
activity in the range 3.0-8.0, the optimal pH for L1 (the isozyme of laccase) was 4.0 while the
optimal pH for L2 was 4.0. 5.0. Laccase from Trametes versicolor has high enzymatic activity
over a wide range of temperature and pH but is optimal at pH 3.0 and temperature 50ºC.
Laccase from Stereum ostrea was most active at pH 6.0 and temperature 40ºC.
Most studies show that a pH range of 4.0–6.0 is suitable for enzyme production [61].
1.1.4.4. The isozymes
An organism can have multiple isozymes of laccase, which differ in their amino acid
sequence and some catalytic kinetics. Molds can produce a variety of isozyme laccases that
differ in both the degree of glycosylation and the composition of the carbohydrate moieties.
T. versicolor has 5 isozymes that differ only in carbohydrate composition, their carbohydrate
composition varies from 10% to 45% compared to the mass of the protein composition [33].
Isozymes can vary considerably in their stability, optimal pH, temperature, and affinity
for different substrates. Furthermore, different isozymes may regulate different roles in the
physiology of different species or within the same species under different conditions [118].
1.1.4.5. The reaction kinetics of laccase
The catalytic activity of the enzyme is characterized by the Michaelis-Menten constant
(Km). This constant of laccase fluctuates over a wide range, in the range of 2–500 µM
depending on the enzyme and substrate origin. The lowest Km value for the substrate is
syringaldazine (a dimer form of two 2,6-dimethoxyphenol molecules linked by an azide
bridge). The affinity for oxygen is less enzyme-dependent and only ranges from 20 to 50 µM.
The higher the enzyme concentration, the greater the enzyme reaction rate. The maximum
velocity (Vmax) when the whole enzyme is bound to the substrate, normally the Vmax value
of laccase varies between 50-300 m/s depending on the source of the laccase [7].
ten
1.1.5. Sources of laccase

Laccase is a very common enzyme in nature, they are found in bacteria, fungi,
and plants.
1.1.5.1. Laccase from plant
Yoshida first described plant laccase in 1883 when he obtained it from the
secretions of the lacquer tree (Rhus vernicifera). Laccase is also manufactured by Acer
pseudopla-tanus. In addition, laccase is present in the leaves of Aesculus parviflora.
Besides, laccase is also extracted from the buds of the green tea plant. After studies
on laccases from Rhus vernicifera, many studies focused on the specificity of
laccases produced by other higher plants such as Zea mays (Corn), Populus
euramericana, Nicotiana tobacco, Lolium perenne and
Arabidopsis thaliana. Plant laccase participates in lignin breakdown or stress
prevention, killing plant fungi. Later, laccase was also obtained from cabbage,
beets, beets, apples, asparagus, potatoes, pears and other vegetables [43], [89].
1.1.5.2. Laccase from mushrooms
Lignin is the main building block of wood and is the most common form of
aromatic cyclic carbon compounds on earth. Lignin strengthens the woody plant
stem containing stomata because it acts as a glue, fastening the cellulose and
hermicellulose chains. Furthermore, lignin forms a barrier to attack by wood-
degrading microorganisms and protects the easily degraded sugar compounds.
Chemically, the polymer heteropolymer lignin is chemically inactive, containing
phenylpropanoid subunits, which are held together by covalent bonds. Due to its
complex structure and strong chemical bonds, lignin is not degraded by hydrolysis
like most naturally occurring polymers. Most yeasts are derived from the major
species of the phylum Basidiomycota and the phylum Ascomycota. The main
function of laccase-producing fungi is to biodegrade lignocellulose and thus
contribute to the carbon cycle in the biosphere [42].
In 1896, laccase was first demonstrated in fungi by Bertrand and Laborde.
Laccase from fungi has been studied and investigated very carefully, especially
laccase from Basidomyces . In addition, fungi such as Ascomyces, Deuteromyces,
11
Basidomyces and fungi capable of degrading ligincellulose such as Agaricus
bisporus, Botrytis cinerea, Chaetomium themophilum, Coprius cinereus, Neurospora
crassa, Phlebia radiate, Pleurotus ostrotus ostreatus, Pycnoporus cinnabarius, T.
versicolor [82].
During laccase production, some Basidiomycota fungi are grown on liquid or solid
media. The highest activity was obtained when cultured in the medium
liquid with species Polyporus sp. and Ganoderma lucidum. The different levels of lignin
degradation of laccase for different wood-based feedstocks depend on environmental conditions
and fungal species. There is no specific evidence on the mechanism of lignin degradation or
removal that each enzyme obtained from different microorganisms has a different mechanism
of action. In plants, laccases affect chemolithogenesis, while in fungi laccases are involved in
many cellular processes, including removal of lignin compounds, spore formation, pigment
production, and lignin formation. fruiting bodies and cause disease in plants. Laccase is mainly
known as an extracellular enzyme but there is also evidence for the occurrence of intracellular
laccase of white rot fungi. where intracellular laccase functions as a precursor to laccase
extracellularly and there was no difference between the two laccases [112].
Lignocellulose decomposition plays an important role in the decomposition of wood
debris. The breakdown of lignin by white-rot fungi involves laccases - enzymes produced
during secondary metabolism. A series of studies have shown that the production of laccases
depends on the selection of new producers with higher lignin degradation efficiency. Fungal
laccases have a higher redox capacity than bacterial or plant laccases (up to 800 mV) and the
processes take place
in their nature have important applications in the field of Biotechnology. Thus, the laccase
obtained from the mold is involved in the degradation of lignin or the removal of phenols. In
addition, it is hypothesized that fungal laccase is involved in the synthesis of
dihydroxynaphthalene melanin [103].
According to the research results of Chhaya et al (2019), when mutating strain F.
icnatum LD-3 with UV light and exposed to EMS, the amount of laccase produced was 3 times
higher than that of the natural strain. In addition, according to this study, when
twelfth
culture of mutant strains, using wheat straw and rice bran, laccase yield increased 2 times
compared to natural strains [17].

1.1.5.3. Laccase from bacteria
Bacterial laccase was first isolated from Azospirillum sp. in 1993 in the rhizosphere
of rice. Subsequently, laccases were detected from various bacteria, such as Gram-positive
bacteria, including Geobacterial, Staphylococcus, Lysinibacillus, Aquisalibacillus , and
Gram-negative bacteria, including Pseudomonas,
Delfia, Enterobacter, Proteobacterium and Alteromonas. The most well-characterized CotA-
laccases are from Bacillus, such as B. subtilis, B. pumilus, B. licheniformis, B. halodurans,
Bacillus sp. HR03…[43].
In addition, according to Guan et al (2018), laccase also exists in many other species
as Streptomyces coelicolor, S. cyaneus, S. psammoticus, S. ipomoea, S.
sviceus, S. bikiniensis, S. violaceusniger, S. lividans, S. lavendulae. In addition, laccase
activity has also been found in a small number of cases on Azospirillum .
lipoferum, B. subtilis, S. lavendulae, S. cyaneus and Marinomonas mediterranea. However,
all laccases or laccase-like proteins found in bacteria are either intracellular or periplasmic,
unlike the laccases in fungi and higher plants, which are secreted in the environment.
outside. Bacterial laccases are not only common in Actinobacter but are also found in ab-
and g-Proteobacteria,
laccase from S. coelicolor [30].
Li et al (2014), isolated fungi from the coastal ecosystems of the Pearl River Delta,
China. Based on their rRNA sequence analysis, 74% of fungi
Ascomycota isolates , 23% were Basidiomycota and only 3% were Zygomycota species.
Of these, about 38% are capable of producing laccase. The best laccase producing strains
were PKU F16 isolated from Cladosporium sp. and PKU F18 isolated from Ascomycota sp.
laccase activity on the intermediate compound ABTS obtained 14.6
U/mL and 10.5 U/mL, after 6 days of incubation [67].
13
1.1.6. Effect of culture conditions on laccase biosynthesis 1.1.6.1. Effect of

temperature Some studies have
shown that the optimum temperature for the production of laccase is 25ºC under
light conditions, but in the dark the required temperature is 30ºC. In general, mushrooms
are grown at a temperature of 25oC-30oC for laccase production which is the optimal
condition. When cultured at temperatures higher than 30ºC the activity of enzymes will
be reduced. However, the fungal strain Steccherinum ochraceum isolated from rotting
wood has high heat tolerance and 3 isoforms of laccase also have optimal activity in the
temperature range of 70oC-80oC. Therefore, temperature greatly affects the ability to
produce laccase of fungal strains
[50]. 1.1.6.2. The influence of environmental pH
To date, not much is known about the effect of pH on laccase biosynthesis, but
when fungi are grown in a medium with optimum pH for growth (pH 5.0), laccase is
production is quite fast. Most reports indicate an initial pH level between pH 4.5 and pH
6.0 prior to culture, but at subsequent stages almost no adjustment is required during
culture.
[7], [8].
1.1.6.3. Effect of metal ions

In 2018, Hernández-Monjaraz et al analyzed the effects of metal salts such as
Fe2+ and CuSO4, as well as of bathophenanthrolinedisulfonic acid (iron chelator) and
bathocuproinedisulfonic acid (copper chelator) on intracellular and extracellular laccase
activity of wild-type and non-pathogenic (rho1:hyg) mutant strains from F. oxysporum f.
sp. lycopersici. The results showed that laccase activity in the intracellular enzyme
fraction of wild-type and mutant strains increased with the addition of Fe2+ waste (53.4%
and 114.32%, respectively). With Cu2+, it was found that there was inhibition of activity
with the addition of CuSO4 for the extracellular enzyme of the wild-type and mutant
strains (82% and 62.6%, respectively) and for the intracellular enzyme of the strain.
mutations (54.8%). The obtained results showed that a different upregulation of
intracellular laccases in the mutant strain compared with the wild type in the presence of
CuSO4 and the copper chelator could be attributed to the rho gene mutation [34].
14
In addition, there are also many studies on the effect that metal ions can
increase or inhibit laccase activity. Specifically, in the environment with Cu2+ Mn2+ ,
and Fe2+ ions will increase the activity of phenol oxidase, especially laccase, while
the presence of Pb2+ and Cd2+ in the environment will inhibit the activity.
of laccase [109].
1.1.7. Application of laccase
Laccase belongs to the blue polycoron oxidase, widely distributed in fungi and
higher plants. It is found in Ascomycetes, Deuteromycetes, Basidiomycetes and is
found in abundance in white rot fungi. With a lot of potential, laccase has been
applied in many fields such as textile, pulp, paper and food industry. More recently,
it is being used in the development of biosensors for the detection and removal of
toxic pollutants, the design of biofuel cells, and medical diagnostic tools. Laccases
are also being used as bioremediation agents because they have been used in the
removal of herbicides and certain explosives from the soil [91]. 1.1.7.1. Industrial
applications
Laccase has great potential to be used as a multi-tasking biocatalyst in the

entire papermaking process. It can participate in pulping, pulp separation alone or
in combination with other bleaching enzymes effectively. Laccase is highly
appreciated for its ability to biotransform pulp fibers, decolorize and stabilize the
effluents of paper mills, and biotransform high-molecular-weight lignin into aromatic
compounds. lower molecular weight. Besides, this enzyme has great potential to
apply to old newsprint bleaching and pulp removal from various pulps [104]. 1.1.7.2.
Application in food industry Residual phenol in fruit juice can cause
turbidity and affect sensory quality
its. Laccase is used to remove phenolic compounds from fruit juices.

Aflatoxin B1 is a mycotoxin that mainly contaminates crops and their derivatives.
It has been reported to trigger the formation of cancer cells as well as cause serious
challenge
15
on human health from consuming contaminated food. Laccase as a useful tool in the degradation
of aflatoxin B1. Studies have shown that due to the
Non-phenolic of aflatoxin B1, the laccase-catalyzed aflatoxin degradation requires a longer time and
is more efficient in the presence of the intermediate. Many reports show that the decomposition
time of Aflatoxin B1 is about 55 minutes to 72 hours. Aflatoxin B1 was reduced by 50% 48 h after
laccase exposure. Furthermore, the use of laccase prevents the presence of chemical residues
after treatment [86].
In wine processing, oxidation of polyphenols increases color and alters flavor, and laccase
helps to remove polyphenols that stabilize wine.
Laccase is also used to remove phenolic compounds that inhibit the fermentation of various species
sugars present in the hydrolysis of lignocellulose [114]. 1.1.7.3.
Application in environmental pollution treatment Dyeing
wastewater has toxic effects on humans and aquatic organisms, causing serious environmental
pollution and ecological risks. According to Wenting Zhou et al (2021), laccase is a promising
biocatalyst for microcontaminant removal and water purification when it is immobilized with some
other compounds. When laccase combines with calcium particles at pH 5, temperature 30oC, in 2
hours will remove up to 99% of Bisphenol A. In particular, laccase from KU-Alk4 is fixed on Cu
atoms, capable of removing 100 % colorant Indigo carmine at 25°C, rotational speed 200 rpm,
for 2 hours [122].
Liu et al (2019) reported that two strains of bacteria, algae and Salmonella marisflavi, isolated
from a marine environment had a higher ability to degrade dye color than other strains isolated from
non-saline sources. [71].
Purified enzymes can effectively degrade synthetic dye colors at pH 6.2 and pH 9.0. According
to many research results, Indigo blue color was almost completely lost within 1 hour and about 93%
of Reactive black color was lost after 1 hour.
Besides, the chlorine separation rate improved to 80% and 97% after 6 hours incubation, at
pH 6.2 and pH 9.0. In addition, the laccase from S. coelicolor was also shown to be able to
similar function, they are able to decompose Black lipstick and Indigo color at pH
9.0, after 1 hour [22].
16
The soil for growing crops, sometimes with high salinity due to irrigation or
from the inappropriate use of chemical fertilizers, herbicides and pesticides. High
salinity can make biodegradation more difficult because it negatively affects
microbial growth and activity. According to a study by Kadri et al. (2017), laccases
from some Basidiomycota fungi , Dacryopinax elegans isolated from decayed
wood in Brazil, were able to degrade diuron compounds in herbicides in the
presence of NaCl. Polycyclic aromatic hydrocarbons (PAHs), aromatic
hydrocarbons with two or more benzene rings, etc. are toxic, carcinogenic,
mutagenic, and non-biodegradable pollutants. The majority of PAHs come from
human activities, including the incomplete combustion of organic matter such as
fossil fuels, coal tar, wood, garbage, oil spills, etc. In soil, PAH often binds to soil
particles and is difficult to degrade by bacteria. The
Intracellular and extracellular enzymes play important roles in the biodegradation
of PAHs, such as cytochrome P450, MnP and laccase. Laccase from white rot
fungus is capable of oxidizing PAH to the corresponding PAH quinone and finally
to CO2. Laccase obtained from Fusarium solani - isolated from mangrove
sediments in Hong Kong, can degrade anthracene and benzene anthracene, can
use anthracene and benzene anthracene as a sole carbon source. After 40 days,
F. solani could remove 40-60% of anthracene and benzene anthracene [47].
1.1.7.4. Some other
applications Laccase is not only used in food, in the pulp and paper
industry, in the textile industry, in environmental pollution... but also in many other
applications. Laccases catalyze electron transfer reactions without intermediates,
they can also be used as biosensors to detect various compounds such as
phenol, oxygen, azide and biosensor, laccase can be detect
morphine, codeine, catecholamine or other enzymes in fruit juices and plant
flavonoids. Polysaccharides have been widely practiced as enzyme carriers
because they can be readily chemically modified according to the nature of
immobilization. This process improves the stability and shelf life of laccases in
catalytic reactions. In addition, selectivity of enzymes can be conserved for
17
specific application after immobilization to polysaccharide. The incorporation of natural
polysaccharides as support materials for laccase immobilization has expanded the modified
biocatalysts for industrial applications [102].
A biosensor is a device that detects, transmits and records information related to
physiological or chemical, biological changes. Laccase is also used in biofuels and biosensors to
detect various compounds and metabolites. Laccase has high potential in nanobiotechnology to
create highly sensitive biosensors. Laccase also plays a huge role in organic chemistry as a
derivative
for cyclosporins (eg cyclosporin), hormones (eg ÿ-estradiol) and
phytoalexins (eg resveratrol) [63].
Some recent reports showed that the antibiotics doxycycline, chlortetracycline,
oxytetracycline and tetracycline were degraded by laccase. As a result, the enzyme's performance
on the removal of anti-inflammatory drugs depends on the surface compound and the source of
the enzymes. Margot et al (2013), studied and concluded that laccase isolated from T. versicolor
degraded to 95% mefenamic acid and 25% of diclofenac
within 20 hours [78].
According to the study of Yitong Jia et al (2022), a laccase obtained from Trametes hirsuta
MX2 expressed in Pichia pastoris denoted rLac1 has strong decolorization ability against remazol
brilliant blue R, the yield reached 92.57% after 3 hours in the absence of ABTS, but for acid red 1,
crystal violet, neutral red, the decolorization rate was only 15.3%, 14.2% and 12.3%, respectively
under the same conditions. However, in the presence of ABTS, the decolorization efficiency was
significantly increased specifically with remazol brilliant
blue R, acid red 1, crystal violet and neutral red decolorization rate is 99.2% respectively,
67.1%, 38.9% and 52.3% [130].
1.2. PERFORMANCE SYSTEM Pichia pastoris 1.2.1. General
introduction to the P. pastoris system According to the
taxonomy, P. pastoris belongs to the kingdom Fungi, phylum Ascomycota, phylum Ascomycota, and phylum Ascomycota.
sub Saccharomycotina, class Saccharomycetes, Order Saccharomycetales, family
Saccharomycetaceae, genus Pichia, species P. pastoris.
18
They have the following characteristics: living at 30oC- 32oC, pH: 3-7, metabolizing
methanol, glucose, sorbitol, glycerol,...and needing a lot of oxygen in the process of metabolizing sources.
carbon.
According to Kielkopf et al. (2021), P. pastoris has become one of the most widely studied
yeasts, it is reported to be one of the most useful and versatile systems for heterologous protein
expression. This expression system is of particular interest due to the strong inducer ability of the
promoter (pAOX1), the ability to secrete extracellular proteins that are easy to purify, and self-
modifies after post-translational processes including glycosylation and morphogenesis. into high-
performance, disulfide linkage, the genomes of P. pastoris
simple operation. P. pastoris is a single-celled organism, so the use of manipulation techniques at
the cellular level is easy. Besides, they also grow quickly on nutrient-poor environments. Similar to
other eukaryotes, P. pastoris is capable of post-translational protein processing including protein
structural folding, proteolytic processing, disulfide bond formation, and glycosylation.
Yeast P. pastoris is capable of metabolizing methanol as the sole carbon source
its. The potent promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol
and it is used for expression of the gene of interest. Accordingly, the expression of extracellular
proteins can be induced by adding methanol to the medium
growth market [54]. 1.2.2.
Advantages and disadvantages of P. pastoris compared to other expression

systems 1.2.2.1. Advantage
P. pastoris - methyl nutritional yeast, has several advantages over S. cervisiae such as: P.
pastoris has a highly efficient promoter and is tightly regulated by the methanol inducible gene
encoding alcohol oxidase, the first enzyme. of the methanol pathway. In the absence of methanol,
the AOX1 gene is completely deactivated. In addition, the AOX1 promoter responds very quickly
to the addition of methanol in the medium, so it can be said that the AOX1 promoter is an important
factor for the transcriptional control of the cloned gene and the production of large amounts of
recombinant protein. . Furthermore, it is possible to select the time of cloning gene induction for
maximum production of recombinant proteins during large-scale fermentation. A further advantage
of the expression system
19
P. pastoris does not synthesize ethanol, so it is possible to obtain high cell density and
secrete large amounts of protein. In addition, P. pastoris normally secretes very few proteins,
thus simplifying the purification of secreted recombinant proteins. The P. pastoris system
was developed cheaply and quickly, with a high cell density above 400 g/L. Therefore, the
production of a wide range of recombinant proteins that can occur in P. pastoris is superior
to that of any other yeast species [131].
In addition, P. pastoris has some other advantages such as having a post-translational
transformation system; the product obtained is in the form of active coils; the terminal
methionine increases protein stability and reduces antigenicity. For the production of healing
proteins, many of which require post-translational modifications. Economically, when using
P. pastoris , the cost of cleaning is lower than that of proteins from other common strains or
commercial application vectors. There are many reasons why P. pastoris has become a
widely used expression system. The strong induction of the AOX1 promoter can be used for
protein production. The protein secretions in P. pastoris favor the clearance of recombinant
proteins. The expression level of protein in P. pastoris obtained in the bioreactor can be
increased 10-12 fold. Thus, the yield of recombinant laccases in P. pastoris is likely to be
greatly enhanced and will reduce production costs. The yeast P. pastoris is an established
expression protein for the production of industrial, food and feed production enzymes..For
all of the above reasons, the expression system of P. pastoris was chosen for production of
many eukaryotic proteins [49], [54].
1.2.2.2. Disadvantages
In addition to the advantages, the expression system of P. pastoris also has
disadvantages such as: the methanol inducer is flammable and explosive. The secreted
foreign protein is easily degraded by proteases. Long expression times may cause some
recombinant proteins to agglomerate due to their high hydrophobicity or molecular interactions [54].
1.2.3. Homologous recombinant expression system using P. pastoris
1.2.3.1. Expression vector system
There are many P. pastoris expression vectors designed. Basically an expression
vector of P. pastoris consists of: a promoter sequence (usually AOX1 promoter)
20
control genes, P. pastoris AOX1-derived transcription terminators , a transcription
promoter, a selective marker gene in E.coli and a selective marker gene in yeast. The
addition of a signal sequence either from the P. pastoris phosphatase gene PHO1 or
from another yeast facilitates the secretion of a recombinant protein. Most of P. pastoris
vectors are designed as implanted plasmids to stabilize plasmids during growth [55], [60].
The main components of the vector include: Promoter AOX1 that controls gene
expression, ÿ-factor peptide signaling sequence, recognition sequence of restriction
enzymes for gene cloning, c-myc receptor analyzer Expression and tail of 6xHis purified
recombinant protein, gene encoding the antibiotic Zeocin, Ori replication region [123].
Figure 1.6. The pPICZÿ expression vector system A, B, C for P. pastoris
(Invitrogen, USA) [123].
1.2.3.2. Factors affecting the expression of P. pastoris
Temperature Temperature has a great influence on the expression of P. pastoris.
Many studies have suggested that the biggest problem with cell secretion of foreign
proteins is protein folding and that the cell's ability to secrete proteins into the culture
medium is due to increased cell lysis. , causing degradation, proteolysis and
21
Important in the culture process is the culture temperature [11], [41]. When the culture temperature
was decreased, the recombinant protein production rate decreased, the endoplasmic reticulum
was actually slightly stressed, preserved the folding capacity of the endoplasmic reticulum, and
enhanced cell viability [28]. ].
P. pastoris has an optimal growth temperature in the range of 30oC, the minimum
induction stage temperature is 15oC which has been shown to enhance extracellular protein
expression without significantly affecting cell growth. Low temperatures can also improve yield
because of reduced protease activity. When the inducible phase temperature of P. pastoris was
reduced from 30oC to 23oC, the production of a recombinant protein tripled. This result can be
explained by the fact that at low temperature the product stability increases, the cell viability also
increases but the cell viability increases.
protease degradation is reduced. In a study by Jahic et al (2003), also reported similar results, at
low temperature, the yield of a protein increased by 100% in recombinant fusion due to decreased
protease activity. The reduction at lower temperatures is due to decreased protease activity and
no protease production [41].
Culture temperature is an important factor that greatly affects the growth and biosynthesis
of enzymes. Every microorganism has an optimal temperature for their growth.
The optimum growth rate of P. pastoris was 30oC (Pichia expression kit – Invitrogen).
According to many studies, P. pastoris can be propagated at temperatures as low as 15oC,
At this temperature the amount of endogenous protease will decrease and increase the amount of foreign protein.
Several other studies have also shown that low-temperature expression can increase the
recombinant protein content, although the fermentation time is longer at 30oC [35]. *
Effect of pH P. pastoris
is commonly cultured at different pH values from 3-7. If the pH value is outside this range,
it will affect cell growth, possibly affect both protein stability and extracellular protease activity, in
addition may cause salt precipitation. However, if the pH is below 4, it will be detrimental to the
production of laccase. This could be explained by the sensitivity of the protease to acidic
environments. The pH in the culture medium can affect both protease activity and the stability of
expressed protein, so it must be monitored and maintained within an optimal range.
22
Based on research results, the most optimal pH activity for recombinant proteins ranges from
5.5 to 8 to minimize protease activity while maintaining protein stability, higher pH values
reduce mobility and may reduce active recombinant product stability. The protease and
aspartic acid secreted by P. pastoris are activated at low pH values, which may explain the
influence of pH on activity.
proteolytic activity [23].
According to many studies, the optimal pH values of pure laccase for oxidation ABTS,
SGZ, 2,6-DMP is 4.2; 6.2 and 6.6 respectively. The pH stability tests indicate that the
recombinant enzyme has its alkaline resistance. Specifically, at pH 3.0 laccase lost 77.12% of
its activity after 24 h, but retained very high activity at pH 7.0.
and 9.0 (120.37% and 237.73%, respectively) after 10 days of incubation at 30oC
[66]. * Effect of dissolved oxygen concentration
Oxygen is an essential nutrient in the expression of P. pastoris.
During the induction phase it is necessary to minimize the amount of oxygen, as it adversely
affects extracellular protein production. Other reports suggest that some recombinant proteins
are produced with higher yields in oxygen-restricted environments. Bo Wang et al (2018)
developed a predictive model of recombinant protein yield based on temperature, pH and DO
concentration in the culture medium. The optimal conditions for the cultivation of P. pastoris
predicted by this model are at a pH of 5.64 and a temperature of
20.24oC [12].
In a study by Baumann et al. (2010), when applying hypoxic conditions to the
continuous culture of a strain of P. pastoris in an antibody Fab fragment, Pgap increased the
protein production rate 2.5 times. Recombinant. Based on these results, multiple models were
designed to control glucose by maintaining ethanol concentrations around 1.0% (v/v), resulting
in a 230% increase in recombinant protein biosynthesis. [11].
There is no single optimal culture method for recombinant protein production, as the
process is influenced by many factors such as degradation of the recombinant protein,
proliferation of extracellular proteases, or nutritional deficiencies due to recombinant protein
production. methanol causes,.. these are obstacles to the development of large-scale operations, affecting
23
economic efficiency due to the very strict purity requirements of the resulting product.
Recent studies have focused on methods to optimize recombinant yields. For
example, novel 'artificial' promoters can be developed that
comparable or superior activity to pAOX1 without the drawbacks associated with
methanol [51]. * Effect of cell
density Cell density prior to
induction also affects the production of foreign protein products. It is an agent
that strongly influences the survival and growth rate of cells in the population. At low
density, cells grow quickly, efficient use of the medium increases protein biosynthesis.
At high densities, cell density reaches saturation in a short time and rapidly enters
the apotosis cycle. Therefore, in previous publications, the authors used different
densities of input cells, ranging from 0.5 to 5 OD values. Many works have
demonstrated the growth stage of cells. The cell in which the protein is induced has a
major influence on protein synthesis and activity, so cell density should be optimized
prior to the addition of inducers [23], [51]. . * Effect of inducer concentration
One of the factors determining the successful expression of recombinant

proteins in P. pastoris is the concentration of the methanol inducer, because the
expression of foreign genes in P. pastoris is efficiently and tightly controlled by promoter AOX1.
Methanol introduced into the culture medium will be oxidized to hydrogenperoxid
(H2O2) and formaldehyde by alcohol oxidase (AOX) (in P. pastoris cells ). During
expression, however, the methanol level must be kept within a relatively narrow
range. Excess methanol can be cytotoxic and strongly reduce AOX promoter activity,
which can lead to cell death [52].
Therefore, methanol is an important inducer in the expression of recombinant
protein by strain P. pastoris. Protein expression related to the activity of AOX1 and
AOX2 genes. If methanol is induced at too low a concentration, it will interfere with
transcription, reducing the amount of foreign protein produced. Induction of methanol
at high concentrations can also be cytotoxic, inhibiting yeast growth [31].
24
Some microorganisms can use the same inducer substrate for laccase
biosynthesis, but the concentration of the inducer used is completely different. According
to the manufacturer's instructions (Pichia expression kit, Invitrogen), 100% methanol
was added every 24 h to induce foreign protein expression in P. pastoris a final
concentration of 1%. During the shaking culture, the methanol was also evaporated fairly
quickly. Therefore, the inducer concentration was added to different final concentrations
to determine its effect on the expression of laccase.
In addition, the presence of methanol is required for high levels of transcription. It is
recognized that an excess of methanol results in an efficient transcription of AOX1 and
poor cellular metabolism, especially when cells are exposed to high concentrations of
methanol for long periods of time. The concentration of methanol in the induction phase
directly affects protein production and cell growth [81].
* Effect of shaking speed
Shaking speed plays an important role in the growth and development of yeast
strains. The shaking speed contributes to an even distribution of cell density and provides
an adequate amount of oxygen for the cells to use. Therefore, shaking speed greatly
affects the ability of laccase biosynthesis of recombinant yeast P. pastoris strain. Many
studies have shown that shaking speed is the only basis to provide dissolved oxygen for
yeast survival in aqueous medium, affecting protein synthesis and directly affecting
secretion of yeast. extracellular enzymes [11].
The standard approach to foreign protein production in P. pastoris is based on a
strategy of full substrate addition in the growth phases, the extreme oxygen utilization
phase, which is closely related to the shaking rate. . The higher the shaking speed, the
greater the use of oxygen, promoting growth and induction
[81].
1.3. INDUSTRIAL DYES AND TREATMENT MEASURES
1.3.1. Industrial Dyes Dyes are

colored organic substances that strongly absorb certain parts of the visible light
spectrum and have the ability to bind to textiles under specified conditions of color
fixation. Dyes can be of natural origin or
25
synthetic. Currently, people almost exclusively use synthetic dyes. Distinctive features of dyes are
color fastness and non-degradability. The color of a dye is determined by its chemical structure: in
general, the structure of a dye consists of a carrier group and a color auxiliaries. Colored groups
are groups containing conjugated double bonds with non-stationary electrons such as: -C = C -C
=N -, -
N = WOMEN -, -NO2,… Color-supporting groups are electron donor or acceptor substituents such as: -
NH2, -COOH, -SO3H, -OH… play the role of enhancing the color of the chromogenic group by
shifting the energy of the electronic system [18].
Dyes are very diverse in chemical composition, color, and scope of use. There are two
basic ways of distinguishing dyes. By chemical structure: azo dyes, antraquinone dyes, polymethyl
dyes, indigoite dyes, sulfur dyes, arylmethane dyes. According to the way of use: acid dyes,
reactive dyes, base-cation dyes. Synthetic dyes have been around for a long time and are widely
used in textile, paper, rubber, plastic, leather, cosmetic, pharmaceutical and food industries. Dyes
have the characteristics of being easy to use, cheap, stable and diverse in color. However, the
widespread use of dyes and their products causes water pollution affecting humans and the
environment. When entering water sources such as rivers, lakes... with a very small concentration
of dyes, it gives a sense of color. The dark color of wastewater interferes with the absorption of
oxygen and sunlight, which is detrimental to the respiration and growth of aquatic organisms.
Thus, it adversely affects the ability of microorganisms to decompose organic substances in
wastewater. For fish and aquatic species, fish tests of more than 3000 dyes fall in all categories
from non-toxic, moderately toxic, very toxic to extremely toxic. Of which, about 37% of dyes are
toxic to fish and aquatic organisms, only 2% of dyes are very toxic and extremely toxic.
toxic to fish and aquatic life [53]. For
humans, dyes can cause skin, respiratory, and lung diseases. In addition, some dyes or
their metabolites are very toxic and can cause cancer (eg, Benzidine dyes, Sudan). European
manufacturers have discontinued this type, but in fact they are still found on the market
26
due to low cost and high dyeing efficiency. The chemical treatment of materials includes wet treatment
and dry treatment. Wet treatment includes: pre-treatment, bleaching, polishing, dyeing, flower printing.
The wet treatment process uses a lot of water, in general, to complete 1 kg of textiles requires 50,300 liters
of water depending on the type of material and equipment. Most of the water
This size is 88.4% will be discharged, 11.6% of the water evaporates during the machining process [57].
1.3.2. There are a variety of techniques for dye color treatment, such as
chemical, physical and biological treatments, including adsorption, coagulation, flocculation, septal
filtration, and ozonation. , electrochemical, irradiated, bacterial, algae, fungal, enzymatic and higher
oxidation processes have been known to decolorize textile dyeing wastewater.
1.3.2.1. physicochemical method
The physicochemical methods are known to be effective in dyeing wastewater, in which the
adsorption method is of most interest due to its simplicity and low sensitivity to toxic pollutants. In addition,
there are other physicochemical methods to remove dye colors such as cotton precipitation, flocculation.
Electrochemical coagulation is applied to remove colors from solutions containing mixed base colors and
dispersions [48].
1.3.2.2. Advanced oxidation method
This process includes techniques such as color oxidation by Fenton system (solution containing
H2O2 and iron), photochemical ultraviolet (UV) light at normal temperature and pressure.
Advanced oxidation process is widely used to remove dyeing dye wastewater and persistent organic
components present in wastewater. The versatility of the enhanced oxidation method relies on the
generation of OH- free radicals . The methods demonstrate superiority over conventional treatment
methods because they can remove non-biodegradable or non-biodegradable organic compounds [48],
[117].
1.3.2.3. Biological fermentation method
Decolorization of dyes by biological methods is an environmentally friendly solution and promises
to be used a lot in the future. Microorganisms such as bacteria, fungi, algae, yeasts, and molds can be
used to remove color through aerobic, anaerobic or combined anaerobic-aerobic processes. Using
microorganisms for treatment is a relatively inexpensive and environmentally friendly method; However,
the use of
27
They depend on the identification of suitable bacterial strains and the establishment of suitable
conditions for dye color degradation, requiring prior physical or chemical treatment steps.
Furthermore, many synthetic dyes have the ability to inhibit bacterial attack. Therefore, the
use of enzyme method is now considered as the most promising alternative for dye removal
from industrial wastewater [40].
1.3.3. Treatment of dye color with laccase The
method of using laccase is an interesting and potential solution for the removal of dye
colors with diverse and complex chemical structures. Laccase has the ability to decolorize
dyes through a nonspecific free radical mechanism that produces phenolic compounds.
Laccase oxidizes the phenol group of the azo dye, with the participation of an electron to a
phenoxy radical, which is then oxidized with a carbonium ion. The carbon-containing phenol
ring bearing the azo bond is attacked by water to yield 3-diazenyl benzenesulfonic acid (III)
and followed by 1,2-naphthoquinone. Free radicals generated by laccase-induced dye
oxidation tend to react with 1,2-naphthoquinone. The free radicals can undergo oxidation to
form compound VIII, which then produces compound X, or polymerize and further oxidize to
form compound X.
IX (Figure 1.7) [79].
Figure 1.7. Mechanism of enzymatic decolorization and degradation of azo dyes [79].
28
With a wide substrate spectrum, laccase has the ability to participate in the process
of color removal or degradation of many dyes with different structures. However, some
pigments are not easily oxidized or are only partially oxidized because they are too bulky in
structure or their redox potential is too high to bind to the active site of the laccase. In this
case, the mediator is a necessary condition for the laccase to act as a catalyst. Several
compounds containing natural phenol rings, including syringaldehyde and acetosyringone,
have been shown to be suitable binders for laccases to exhibit efficient performance in the
dye category. Although the molecular weight of the degradation products is smaller, the
toxicity is not reduced compared with the original compound [73].
Therefore, the color removal efficiency can be high, but the dye treatment process
still needs to be further studied until it is completely removed from the intermediate products
that are still potentially hazardous to human health. Some applications in the degradation of
substances including phenols, polyphenols, PCBs, PAHs, dyes and azo dyes by laccase are
shown in Table 1.1.
In Vietnam, the filamentous fungus Trichoderma sp. FCP3 was isolated from Cuc
Phuong National Park biosynthesized crude laccase capable of removing some dyes with the
following yield: NY3: 92%; RBBR: 86%; NY5: 64%; NY1: 60%, NY7: 6%
[twelfth].
With the development of science and technology in general and biotechnology in

particular, the use of microorganisms and enzymes from microorganisms to reduce the use
of chlorine in dye color treatment has been favored by many groups of authors. Research
and prove that the technological solution is feasible and brings high economic efficiency.
P. ostreatus and T. versicolor are considered as typical fungi in laccase biosynthesis and
application studies. In addition, other fungi species known to be able to biosynthesize laccase
such as Agaricus bisporus, C. unicolor, Coprinopsis cinerea, Coriolopsis gallica, Cryptococcus
neoformans, Cyathus bulleri, Fomes fomentarius, Ganoderma lucidum, Panus rudis, Phlebia
radiata, P. brumalis, P. cinnabarinus, P. sanguineus, Rigidoporus microporus, Schizophyllum
commune [32], [128].
29
Table 1.1. Application of laccase in dye biodegradation Dyes Source of laccase
Mediator Format
Acid Orange 67, Basic Red 18, Basic Paraconiothyrium variabile HBT Free
Yellow 28, Direct Black 166, Direct
Yellow 107, Disperse Yellow 79
Bromophenol Blue, Congo Red, Laccase re combination - Free
Coomassie Blue, Tripan Blue Trichoderma sanguineus
expressed in Trichoderma
atroviride
Acid Black 172, Congo Red, Crystal Trichoderma pubescens - Free

Violet, Direct Fast Blue FBL, Indigo
Blue, Naphthol, Green B, Methylene
Blue, Neutral Red, Reactive Brilliant
Blue X-BR, Remazol Brilliant, Blue
Reactive (RBBR)
Brilliant Blue G, Brilliant Blue R, Pycnoporus sanguineus VA Free
Bromophenol Blue, Coomassie Blue
R250, Crystal Violet, Malachite
Green, Methylene Blue, Xylene
Cyanol, RBBR
RBBR Trametes versicolor HBT Permanent
RBBR Cerrena sp. HYB07 - Permanent
Coomassie Blue R250 Cerrena sp. HYB07 ABTS, AS, Free

HBT, SA, YA
The ability to colorize some representative mushrooms has been reported in recent
decades. Among the studied mold strains, the fungus T. versicolor
2008001 with laccase activity of 20,000 U/L removed 98% of color 25 Reactive Blue 49 at
conditions pH 2.95; initial color concentration was 55.6 mg/L after 46.91 min. The laccase-
producing fungus P. ostreatus with 32,450 IU/g activity was able to remove over 80% of the mixture of 5
color Methyl orange, Trypan blue, Ramazol brilliant red, Ramazol brilliant blue and Ramazol
brilliant yellow; when using a mixture of 3 fungi P. chrysosporium, T.
30
versicolor and Bjerkandera sp. BOL13 to remove the dye color of the azo group then
strain Bjerkandera sp. BOL13 removed 89% of the color Remazol Red RR after 13 days [137].
The process of color removal by fungal strains needs to distinguish whether the cause is
due to adsorption, decomposition or enzyme catalysis. The activity of fungal strains rarely leads
to dye mineralization because the mechanism of mineralization is highly dependent on their
chemical structure. Mineralization occurs more strongly in dyes containing easily substituted
aromatic rings than in non-substituted aromatic rings. Better mineralization occurs under nitrogen-
poor conditions. Some other studies also concluded, the ability of fungal strains to use dye as a
carbon source, then the bonds in the dye molecule will be severed and used by microorganisms
as a carbon source. This usually occurs in living microbial communities. Decoloration of three
synthetic dyes using direct F. oxysporum cultures was performed at concentrations of 50 mg/L
and 100 mg/L. The highest decolorization rate of laccase in 3 pigments: Malachite green (98%
and 96.8%); Congo red (95% and 85%) and Methyl orange (87.6% and 83%). The laccases
were able to degrade synthetic dye pigments against Caco-2 and fibroblast cells, as determined
by the Tetrazolium bacterial assay. In addition, the highest decolorization was identified with
Malachite green showing up to 100% cell viability. The results demonstrated that the
decolorization ability of the enzyme laccase
bring high efficiency [129], [136].
The decolorization ability of recombinant laccase has also been studied extensively
Research by Lu et al. in 2009, laccase isolated from Pycnoporus sanguineus and expressed in
the strain P. pastoris SMD1168H was investigated under conditions such as culture temperature
of 30oC, pH 6.5, CuSO4 concentration of 0. 5 mmol-1 and a methanol concentration of 0.5%,
the recombinant laccase decomposed almost completely 4 dyes, azo,
anthraquinone, triphenylmethane and indigo [75].
Research results of Huy et al., in 2021, concluded that the gene encoding the enzyme
laccase (rLoLacc5) obtained from F.oxysporum HUIB05 was expressed in P. pastoris with
optimal culture medium at pH 6.5; At 35oC, shaking speed 210 rpm and Cu2+ ion concentration
is 0.08 mM, rFoLacc5 removed other synthetic dyes.
thirty first
as Brommothymol blue, Methylene blue, Methyl orange, Remazol brilliant
blue R, Aniline blue, Evans blue, Indigo carmine and Orange II [40].
According to the study of Siqi Liu et al (2020), the lac gene from Aspergillus sp. expressed
present in P. pastoris removed up to 45.5% of Congo red pigment [106].
According to research by Ahmet Tülek et al. (2020), the laccase gene isolated from
Madurella mycetomatis (MmLac) was expressed in P. pastoris. MmLac exhibits good activity in
the pH 4.0–6.0 range; At high temperature of 50oC - 60oC, recombinant MmLac enzyme has
the ability to increase whiteness, and exhibits high efficiency and stability. Therefore, this
recombinant enzyme is used in textile biotechnology applications, as well as in food and
environmental technologies [6].
According to the study of Tamara Kyzeková et al., 2020, with high expression level,
growth of cell density and post-translational modifications, P. pastoris is a suitable microorganism
to produce recombinant enzyme . Expression products from P. pastoris can degrade pesticides
or replace them with environmentally friendly substances, the ability to degrade phenolic
compounds, synthetic dyes,
hydrocarbons, antibiotic residues, lignin, nitroaromatics, toluene, bioplastics and toxic compounds
such as cyanide. Its products are also used for pulp bleaching, pollutant detection and medical
treatment [110].
32
Chapter 2. RESEARCH SUBJECTS AND METHODS

2.1. RESEARCH SUBJECT 2.1.1.
Subjects: Mold
strains isolated from plant residues with characteristics of saprophytic mycelium
growing in different locations such as parks, Khai Dinh mausoleum area,... in Thua Thien
Hue province. (Figure 2.1).

The time of specimen collection was conducted in the rainy season when the
mycelium flourished.
2.1.2. Collect samples
Select rotten tree trunks with white rotten fungal populations, use a clean spoon or
knife to scrape this bark into a sample storage bag, take soil from the junction between the
rotten wood and the soil into the sample storage bag and close the bag. Collected samples
were stored at 4oC until isolated cultures.

2.2. RESEARCH METHODS
2.2.1. Isolation of mold strains with laccase activity 2.2.1.1.
Isolation of molds from specimens
Weigh 1 g of the plant sample into a conical flask containing 9 ml of sterile distilled
water, shake well, and settle the residue by leaving the triangle in peace for 5 min. Then
aspirate 1 mL of the supernatant through a conical flask containing 9 mL of sterile distilled
water and mix well by ring shaking. Continue doing so to get 10-3 and 10-4 dilutions .
Then, aspirate 100 µL of sample at the dilutions onto the surface of the previously prepared
agar plate, and spread the sample evenly over the surface of the agar plate with a sterile
swab. Each dilution was inoculated into 3 petri dishes. Then carefully pack and raise in an
incubator at 30ºC for about 3-4 days. Select characteristic colonies and grow separately
and transfer to new PDA agar, also cultured at the same temperature. After the seeds
grow well and are pure, they are stored in cold conditions (4oC - 8oC) to keep seeds [126].
2.2.1.2. Screening for laccase biosynthetic mold strain
The isolates were transferred to BSM (basal solid medium) screening medium
supplemented with 0.01% guaiacol as an indicator as described by Xu et al., (2015). Using
a sterile loop inoculation, block a piece of agar about 1 cm2 with fibers
33
fungi on potato agar extract (PDA), transfer the agar plate to a plate of BSM screening medium for
the mycelium in contact with the medium. Incubate at 30ºC for 24 days. Visually, a reddish-brown
ring around the colony was formed on the medium supplemented with guaiacol to detect the fungal
strain.
ability to produce laccase. Visually, the darker the red-brown ring, the stronger the laccase activity.
Fungal strains with laccase activity were collected and stored at -80oC and cultured to check
extracellular laccase activity [126]. 2.2.1.3. Biological characteristics of mold strains
Plate agar PDA medium was used to study the morphology of fungal strains. After 3 days of
culture at 30oC, when the fungi had grown strongly, they were observed directly with the naked eye
and observed under an optical microscope. Observe the morphological characteristics of colonies:
growth, development, color, shape and surface of colonies, secretion of droplets, pigment...
Observation of microscopic characteristics: characteristics of mycelium, sporophyte and sporophyte
formation, fungal sporulation characteristics using a Nikon optical microscope (Eclipse 55i, Nikon,
Japan) with x40 magnification [3].
2.2.2. Investigation of laccase biosynthesis from isolates
After being selected from the previous experiment, the fungal strains were selected for
laccase biosynthesis culture on BSM minimal mineral medium containing straw or wood substrates
with material size ÿ 2 mm. The fungal strains grown on agar plate containing PDA medium were
transferred to a conical flask containing 20 mL of liquid potato extract (PD) medium and cultured at
30ºC, 180 rpm, for 3 days [84].
After having mycelial biomass, use a micropipette to aspirate 1 mL of the transferred biomass
into a conical flask containing 50 mL of enzyme-producing medium consisting of:
- Semi-solid straw medium (symbol: MF1): BSM medium (not added
glucose and agar), supplemented with 10% straw powder.
- Semi-solid wood medium (symbol: MF2): BSM medium (not added
glucose and agar), supplemented with 10% wood pulp.
- Liquid wood medium (symbol: MF3): BSM medium (without glucose
and agar), supplemented with 5% wood pulp.
34
- Liquid straw medium (symbol: MF4): BSM medium (without glucose
and agar), supplemented with 5% straw powder.
Each strain cultured 3 flasks on each medium at 30ºC for 18 days (shaking speed 180
rpm for liquid fermentation). Every 3 days, 1 mL of culture was collected and enzyme activity was
determined to determine the suitable medium for laccase biosynthesis of mold strains [84].
2.2.3. Determination of laccase enzyme activity
The laccase activity was determined based on the oxidation of ABTS (2,2'-azino-bis 3-
ethylbenzothiazoline-6-sulfonic acid) to form a strong light-absorbing compound.
at 420 nm [126]. An enzyme-containing solution was incubated with 1 mM ABTS in 100 mM
sodium acetate buffer, pH 4.5 in a reaction volume of 1 mL. The reaction was carried out at
annealing temperature of 40ºC for 10 min. Then, the reaction solution was measured at 420 nm
(ÿ420 = 36.0 mMÿ1/cm) by spectrophotometer.
U-2910 spectrophotometer (Hitachi, Japan) [84].
One unit of laccase activity is the amount of enzyme required to form 1 µM product from
ABTS in 1 min, under experimental conditions.
2.2.4. Methods for the determination of extracellular
proteins The protein concentration in the sample was calculated based on a calibration
curve with a standard protein solution of known concentration. The standard protein solution was
bovine serum albumin (Nacalai, Japan), diluted to concentrations of 1.25; first; 0.75; 0.5; 0.25 µg/
mL. After adding the protein solution to the reagent solution (20 µL of standard enzyme solution/
980 µL of Bradford 1X reagent), the color appeared after 2 minutes and persisted for up to 1 hour.
Measure the optical density of the solution with a spectrophotometer at wavelength .
595 nm [59].
Two kinetic parameters, Michaelis constant Km and maximum reaction rate Vmax, were
determined through the reaction between laccase and ABTS substrate at concentrations of 1
mM, 2 mM, 4mM, 6 mM, 8 mM, 10. mM and 50 mM. The reaction mixture consisted of purified
laccase and ABTS substrate at different concentrations and sodium acetate 100 mM, pH 4.5,
incubated at 45ºC for 60 min. The reducing sugar product produced in the enzymatic reactions
was calculated as the amount of ABTS according to the calibration curve.
35
A graph of the dependence of the reaction rate (V) on the substrate concentration (S) is plotted.
Determine the dependence between 1/V and 1/S, from which the first order Lineweaver Burk
equation is established as:

first
= first
+
first
or y = ax + b accordingly determine
[]
first
get kinetic parameters of enzyme with substrate: Vmax = and Kmax= . In there
V is considered as the number of micromoles between laccase and ABTS released in 1 min, [S] is
calculated as ABTS concentration (measured in mM/ml).
(This part Thoa sent, but there is no reference - Please help me check)
2.2.5. Determination of some characteristics of crude laccase from isolate
2.2.5.1. Effect of pH
To find the optimal pH for the laccase catalysis reaction, aspirate 10 µL of the enzyme
incubated with 100 µL of acetate buffer (including CH3COONa and CH3COOH) with different pH
levels: 3, 4, 5, 6, 7 and 8, adding 10 µL ABTS and add sterile distilled water so that the reaction
volume is 1 mL. The mixture was incubated for 30 min, then the OD was measured at 420 nm to
determine the laccase activity. Proceed similarly for the control sample (10 µL of inactivated enzyme
incubated at 100°C, for 10 min) [84]. 2.2.5.2. Effect of
temperature Temperature level with
the 20ºC mark; 25ºC; 30ºC; 35ºC; 40ºC; 45ºC; 50ºC was used to test the effect of reaction
temperature on enzyme activity. The reaction conditions were similar to those described in section
2.2.5.1 [84]. 2.2.5.3. Effect of metal ions
Metal ions including Fe2+, Cu2+, Ca2+, Mn2+, Mg2+ , Zn2+, Co2+ were used
to evaluate the effect on enzyme activity. Metal ions were added to the reaction to
a final concentration of 5 mM. The reaction conditions were the same as described
in section 2.2.5.1 [84].
2.2.6. Molecular identification of isolated fungal
strains 2.2.6.1. Total DNA extraction
Total DNA of the fungal strain with the strongest extracellular laccase activity was extracted
based on cetyltrimethyl buffered total DNA extraction method
36
ammonium bromide (CTAB) as described by Sambrook et al. (2006) [101] with some minor
adjustments.
The mycelium was grown in 5 mL of PD medium for 3 days at 30°C. Mushroom biomass
was recovered by cold centrifugation at 4°C for 5 min at 14,000 rpm.
Then rinse with sterile distilled water to completely remove the culture medium and resuspend in
500 µL CTAB buffer (100 mM Tris-HCl, pH 8; 1.4 M NaCl; 20 mM EDTA; 2% CTAB ; 0.2% ÿ-
mercaptoethanol). Cells were disrupted by ultrasonic waves at 60 Hz, at intervals of 6 seconds for
5 minutes by ultrasonic device VC-130 (Sonics, USA) combined with incubation at 65°C for 30
minutes. Cell extracts containing total DNA were recovered by cold centrifugation at 4°C for 5 min
at 14,000 rpm. Total DNA was extracted and purified using 500 µL phenol:chloroform:isopropanol
mixture (25:24:1 ratio), mixed by vortex, and separated by cold centrifugation at 4°C for 10 min at
high speed. 14,000 rpm. Aspirate 400 µL of the upper phase mixture, transfer to a fresh 1.5 mL
tube, and total DNA is precipitated with 2 times the volume of pure ethanol, wash the precipitate
with 70% ethanol, and dissolve in 50 µL sterile distilled water. Check electrophoresis: run
electrophoresis on 1% agarose gel supplemented with RedSafeTM
Nucleic Acid Staining Solution 20000x (iNtRON Biotechnology, Korea) by direct current at
80 V for 55 min. The gel is observed under the lamp
ultraviolet (UV transilluminator, Wealtec).
Determination of total DNA concentration: using spectrophotometer method.
2.2.6.2. PCR amplification of ITS 1-4 . sequences
For molecular identification of the isolate, the nucleotide sequence of the conserved region
ITS1-4 was amplified and used primers ITS1 (TCC GTA GGT GAA CCT GCG G) and ITS4 (TCC
TCC GCT TAT TGA TAT GCG G). 77). PCR reaction was carried out at a volume of 25 L
containing 40 ng of total DNA, 10 pmol ITS1, 10 pmol ITS4, 1X
GoTaq® Green Master Mix (Promega, USA).
PCR cycle was performed: denatured DNA at 95°C for 10 min; 30 cycles with 95°C for 60
seconds, 55°C for 30 seconds and 72°C for 60 seconds; for the final cycle lasting at 72°C for 10
min. Electrophoresis to check PCR products on gel
37
agarose 1% in TAE (Tris acetic acid EDTA) buffer at 80 V for 40 min using RedSafe™ dye.
2.2.6.3. Sequencing and building a phylogenetic tree
PCR products were purified and nucleotide sequenced through Firstbase (Malaysia).
The sequence was completed using BLAST on NCBI to evaluate the similarity of the ITS1
conserved region, thereby finding the most similar species with the sequence to be studied.
Methods of building a phylogenetic tree: The laccase gene family tree of the fungal
strain was compared and analyzed using MEGA X software with 500 times boostrap
repetition [62].
2.2.7. Isolation of genes encoding laccase
The isolate with the highest laccase activity was used as the raw material
to isolate genes coding for laccase.
2.2.7.1. Total RNA extraction The
fungal strains with strong laccase activity were grown in 50 mL of PD medium at
30oC, shaking at 180 rpm for 3 days, then transferred the mycelium biomass to BSM
medium and cultured at 30oC. , shake 180 rpm for 6 days to induce
laccase production.
The post-induced fungal biomass was collected by centrifugation, removed the
supernatant, and washed the mycelium twice with distilled water. Total RNA of mycelium
was extracted using Sepasol-RNA I Super G kit (Nacalai Tesque, Japan). Conduct
electrophoresis to check the total RNA obtained on 1% argarose gel, voltage of 80 V for 30 minutes [101
2.2.7.2. cDNA biosynthesis
The cDNA biosynthesis was performed using the RevertAid First Strand cDNA
Synthesis kit (Thermo Scientific, USA). The reaction composition consisted of 5 µg total
RNA; 0.5 µg oligo (dT)18 primer; 4 L reaction buffer (5×), 20 U ribonuclease inhibitor
RiboLockTM, 2 L 10 mM dNTP mix and 40 U M-MuLV RT reverse transcriptase, add sterile
distilled water to total reaction volume is 20 µL. The obtained products continue to perform
PCR amplification.
2.2.7.3. Amplification of the laccase . gene
38
The primer sequences Folac1F, Folac1R, Folac2F, Folac2R, Folac3F, Folac3R and
Folac4F, Folac4R (table 2.1) used to amplify the entire length of the Folac1, Folac2,
Folac3, Folac4 genes were designed based on the same gene in F. oxysporum . f. sp.
lycopersici 4287 was registered at NCBI with code XM_018393180 using FastPCR
software. PCR reaction composition for Folac1 gene: 40 ng cDNA, 6 µL Go Taq® Green
Master Mix 2x (Promega, USA); 0.5 µL forward primer (10 pmol/µL); 0.5 µL reverse primer
(10 pmol/µL) and sterile distilled water to obtain a total volume of 12 µL. PCR reaction
composition for Folac2, Folac3 and Folac4 genes: 40 ng total DNA, 6 µL Go Taq® Green
Master Mix 2x (Promega, USA); 0.5 µL forward primer (10 pmol/µL); 0.5 L reverse primer
(10 pmol/µL) and add sterile distilled water for a total volume of 12
µL.
PCR amplification of Folac1, Folac2, Folac3 and Folac4 genes was performed
present in a heat cycler (Veriti ® 96-well Thermal Cycler, AB Applied Biosystems, USA)
with a thermal cycle: denaturation at 95oC for 5 minutes; 30 heat cycles with each of 95ÿ
for 1 min, 55ÿ for 1 min and 72ÿ for 1 min 30 seconds; finally 72ÿ for 10 minutes. PCR
products were checked by electrophoresis on 1% agarose gel
80 V for 30 minutes.
Table 2.1. Nucleotide sequences of primers used to amplify genes Folac1, Folac2,
Folac3, Folac4
bait Sequence
Folac1F 5'-ATGACGAAGCTATCCCTGACAC-3'
Folac1R 5'-TCAAACACCAGAATCGTCCTGG-3'
Folac2F 5'-ATGGCTCTCATAGAGCGAGATATG-3'
Folac2R 5'-TTAAATACCAGAATCACCTTCAAAG-3'
Folac3F 5'-ATGGTCGCTCACGATGAGAAG-3'
Folac3R 5'-TTACTCACATAACTCCGCCG-3'
Folac4F 5'-ATGTGGATTACAGACGCCTGC-3'
Folac4R 5'-TTAGATACCCGAGTCATCCTGGA-3'
2.2.7.4. Attach the PCR product to the cloning vector
39
PCR products were attached to pGEM®T-Easy vector (Promega, USA) using T4
DNA Ligase (Promega, USA), binding reaction components include: 50 ng of pGEM®-T Easy vector, 5
µL of buffer, 5 units of T4 DNA ligase, 20 ng of PCR product, then add sterile distilled water To obtain a
final volume of 10 L, the binding reaction was incubated at 25 C .
For 1 hour.
2.2.7.5. Transformation into E. coli TOP10 and select the transformed variant
The binding reaction mixture was transformed into E. coli TOP10 cells by heat shock method.
The transform was cultured in a petri dish containing LB medium supplemented with
add 50 g/mL ampicillin, 20 L 5-Bromo-4-Chloro-3-Indolyl –D
Galactopyranoside (X-gal) 50 mg/mL and 100 L Isopropyl ÿ-D-1-
thiogalactopyranoside (IPTG) 0.1 M for 16 h at 37°C.
Check the presence of gene fragment in the recombinant vector pGEM®T-Easy by PCR method
directly from white colonies with specific primer pairs as shown in Table 2.1. Carrying out PCR with the
same thermal cycle as running PCR to amplify Folac1, Folac2, Folac3, Folac4 genes. Electrophoresis
to check results and select E. coli cells carrying recombinant vector. E. coli cells carrying the recombinant
vector were then multiplied by biomass to separate plasmid DNA using the GeneJET Plasmid Miniprep
K0502 kit (Thermo scientific, USA). Conduct EcoRI cleavage of recombinant plasmids to verify
stream generation again.
2.2.7.6. Decoding and sequencing of laccase . genes
The recombinant plasmid carrying the laccase gene was sent for sequence analysis to determine the
Gene nucleotide sequencing at First BASE (Malaysia). The laccase gene sequence was analyzed and
compared with the laccase gene on the world gene bank database
GenBank (https://www.ncbi.nlm.nih.gov). Characterization of the laccase gene was performed using the
free software of the Center for Biological Sequence Analysis, Technical University of Denmark (http://
www.cbs.dtu.dk/index.shtml) and the software. Phyre V2.0.
2.2.8. cFolac1 expression in Pichia pastoris
The DNA fragment carrying the protein coding sequence of Folac1 (symbol cFolac1) was
amplified from the recombinant pGEM-T Easy plasmid Folac1. Pair of primers with the symbol
cFolac1F and cFolac1R with nucleotide sequences as shown in Table 2.2. Pair of primers
40
designed with additional recognition sequences of the restriction enzymes EcoRI and XbaI to
facilitate separation and processing in the following steps.
Table 2.2. Sequence of primers used to amplify the cFolac1 gene from cDNA
bait Expression primer sequence
cFolac1F 5'-GAATTCTTGCCCAAGATGGGTT-3'
cFolac1R 5'-TCTAGACAAACACCAGAATCGTC-3'
cFolac1 was amplified by PCR with a specific primer pair cFolac1F/ cFolac1R. PCR
reaction components: 40 ng of plasmid DNA carrying the full sequence of Folac1 gene, 6 µL Go
Taq® Green Master Mix 2x (Promega, USA); 0.5 µL forward primer (10 pmol/µL); 0.5 µL reverse
priming (10 pmol/µL) and add an amount of sterile distilled water to give a total volume of 12
µL. The specific PCR product was cloned back in with the procedure described in sections
2.2.7.3 and 2.2.7.4. The recombinant GEM®T-Easy plasmid carrying the cFolac1 gene was
extracted and the nucleotide sequences were sequenced as described in section 1.

2.2.7.5.
2.2.8.1. Cloning in the expression vector pPICZÿA
Folac1 was isolated from the recombinant pGEM-T Easy vector by two restriction
enzymes EcoRI and XbaI, followed by electrophoresis on 0.8% agarose gel, purified using
GeneJET Gel Extraction kit K0691 ( Thermo scientific, USA) and attached to the vector
pPICZÿA were also treated for linear angiogenesis with the same two enzymes. The binding
reaction components include: 4 µL pPICZÿA vector, 4 µL laccase gene DNA, 1 µL binding
buffer (10X) and 1 µL T4 DNA ligase. The binding reaction mixture was incubated overnight at 16°C.
The recombinant plasmid pPICZÿA - cFolac1 was transformed into E. coli TOP10 variable
cells . The transformed solution was cultured on a petri dish containing concentrated LB medium
supplemented with 25 ÿg/mL zeocine and incubated overnight at 37°C. Single colonies were
transferred to test tubes containing 5 mL of liquid LB medium containing 25 µg/mL zeocine at
37°C, shaking at 200 rpm for 12 h. Then, the culture fluid was collected and the recombinant
plasmid pPICZÿA/cFolac1 was extracted from E. coli cells using the GeneJET Plasmid Miniprep kit.
41
K0502 (Thermo scientific, USA) and checked by PCR using primers AOX1 and primers cFolac1F/
cFolac1R.
Table 2.3. Sequences of primers used to amplify AOX1F and AOX1R . genes
bait Nucleotide sequence
AOX1F 5'- GACTGGTTCCAATTGACAAGC-3'
AOX1R 5'– 'GCAAAGGCATTCTGACATCC–3'
cFolac1-MORE 5'-CAT CCG CCT CTG TTC TCT GCT-3'
Three recombinant E. coli colonies were randomly selected and plasmid DNA separated
to determine the nucleotide sequence of the recombinant pPICZÿA/cFolac1 prior to transformation
into P. pastoris. For sequencing, primer pairs AOX1F and AOX1R were used as the instructions
of the manufacturer of the P. pastoris expression kit (Invitrogen, USA). In addition, a primer
cFolac1-M was also designed to amplify the nucleotide sequence in the nucleotide region that
the sequence obtained from the primer pair AOX1F and AOX1R did not cover.
2.2.8.2. Transformation into P. pastoris X33
The recombinant plasmid pPICZÿA/cFolac1 was electrically transformed into P. pastoris
X33 cells after being treated with PmeI to generate linear DNA. The transformation reaction
consisting of 80 µL of P. pastoris X33 cells and 10 µL of recombinant pPICZÿA/cFolac1 plasmid
solution were placed in 0.2 cm cuvettes and placed on ice at 4°C for 5 min. Place the cuvette in
the “Shock Pod” chamber of the transformer and run with a 1.5 kV pulse for 5 ms. Cuvettes were
removed from the chamber, immediately added 1 mL of cold 1 M sorbitol, then transferred the
cuvette mixture to a 15 mL tube and incubated at 30°C for 1 h. Then, the transformation solution
was inoculated onto a petri dish containing YPDS medium (1% yeast extract, 2% peptone, 2% D-
glucose, 1 M sorbitol), supplemented with
supplemented with 100 ÿg/mL zeocine and incubated at 30°C for 24 h.
Check for gene presence in single colonies of recombinant P. pastoris X33: culture these
single colonies in YPD medium and perform total DNA extraction of recombinant P. pastoris X33,
then that checks for the presence of
42
laccase genes by PCR using primer pair AOX1 and gene primer cFolac1 from this total DNA
template.
2.2.8.3. Laccase gene expression
Expression induction was performed according to the recombinant expression
procedure recommended by the manufacturer P. pastoris X33. Select 10 single colonies of
recombinant P. pastoris X33 on YPDS medium containing zeocine and place in test tubes
containing 5 mL of YPD medium supplemented with 100 ÿg/L zeocine and culture at 30oC,
shake 250 rpm for 24 hours . Cell biomass was obtained by centrifugation at 1500 × g for 5
min at room temperature, discarding the supernatant and adding 100 mL of YP medium
supplemented with 1% glycerol. Continue culture at 30oC, shake 250 rpm for 24 hours. Cell
biomass was obtained by centrifugation at 1500 × g for 5 min at room temperature, removing
the supernatant and resuspending with YP medium. Determine the appropriate dilution factor
to add to the 20 mL culture volume such that OD600 = 1. Expression was induced by adding
methanol at OD600 = 1 and maintaining a 1% methanol concentration for 3 days. culture.
2.2.8.4. Determination of recombinant enzyme
activity P. pastoris X33 cell biomass after 3 days of induced culture was removed
from the culture solution. This culture was used to determine laccase activity from recombinant
strains and was carried out according to the method described in section 2.2.3 with a reaction
volume of 100 µL and reaction time. is 30 minutes.
2.2.9. Investigation of factors affecting the expression of recombinant laccase
The extracellular enzyme-expressing P. pasoris X33 cell line was selected to optimize
extracellular enzyme production by investigating several factors such as induction time,
inducer concentration, cell density, and temperature. culture and shaking speed.
2.2.9.1. Culture time
Recombinant P. pasoris X33 yeast cells were cultured to induce enzyme production
on YP medium supplemented with 1 mM CuSO4, supplemented with methanol at OD600 =
1 and maintained at a methanol concentration of 1. % in 5 days. Collect culture samples at
24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours to determine enzyme activity
43
extracellular laccase over time. Experiments to evaluate the expression of laccase were designed
based on the results of induction time studies [116].
2.2.9.2. Inducer concentration
To determine how methanol concentration affects the induction of recombinant laccase
protein expression, the optimally transformed strain was cultured in YP medium supplemented with
methanol for 144 h. The study flasks were supplemented with methanol at concentrations of 0.5%,
1%, 1.5%, 2%, 2.5% and 3% after every 24 h of culture. Expression cultures were obtained for
enzyme activity determination [4].
2.2.9.3. Cell density After
determining the appropriate time and methanol concentration, the optimal conditions were
selected to continue investigating the initial cell density. The cell density at the time of induction
was selected with an OD600 value from 0.5-2. After induction, cultures were collected for enzyme
activity determination every 24 h [44].
2.2.9.4. Culture temperature
To investigate the effect of culture temperature on the production of extracellular recombinant
laccase of strain P. Pastoris X33, single colonies of P. pastoris X33 were cultured with initial cell
density and culture time. , methanol concentration and optimal culture density in the temperature
range from 25oC to 35oC. Expression cultures were collected for enzyme activity determination
every 24 h.
2.2.9.5. Shaking
speed From the survey results, the appropriate culture and induction conditions including:
culture temperature, culture time, methanol concentration, the influence of shaking speed were
investigated with a value of 150 rpm/ min, 180rpm, 210rpm, 240rpm. Recombinant P. pastoris X33
cells were cultured and induced enzyme production as described above. Expression cultures were
collected for enzyme activity determination every 24 h.
2.2.10. Purification of recombinant enzymes
The recombinant rFolac1 protein after induction is recovered by centrifugation
13000 rpm for 15 min at 4oC, purified on a HisTrap FF column (GE Healthcare, USA). Before use,
the HisTrap FF column was washed and equilibrated with the mixture
44
Combination: 5 water (CV) fractions and 1 0.1 M CuSO4 fraction. For the purification step, run 5
fractions with binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole, pH 7.4).
Then, a mixture of extracellular protein and 10x bidding buffer (9:1 ratio) was added to the column
at a flow rate of 3 mL/min. The column is rinsed with the binding buffer. Proteins that do not adhere
to the column are washed away. Finally, the purified recombinant protein attached to the column
was recovered with elution buffer (20 mM sodium phosphate buffer, 0.5 M NaCl, 0.5 M imidazole,
pH 7.4). Proceed to collect each fraction, 1 mL each. The recovered enzyme was used for SDS-
PAGE electrophoresis. Protein content and enzyme activity were determined after each fractionation
along with the recovery efficiency and purity of the recombinant laccase product according to the
following equations:
[40]:
The formula for calculating the recovery yield (%) = Enzyme activity after purification/Activity
enzymes present in the medium. (CT1)
Enzyme activity after purification = specific activity x collection volume (CT2)
2.2.11. SDS – PAGE . Electrophoresis
The gel sheet is poured in two layers, the lower layer is seperating gel (12%) is poured 1.5
cm from the top surface, frozen for 30 minutes, combed and then poured over the top layer of
stacking gel (5%), left for 30 minutes. The gel is completely frozen and stable. The gel was then
inserted into the BioRad electrophoresis system and run. Approximately 4 µg of recombinant
rFolac1 was mixed with 15 µL loading dye 2X. The mixture was put into SDS-PAGE gel
electrophoresis with 5% stacking gel and 12% dissolving gel. First run at 100 V for 30 minutes
(when the sample runs out on the stacking gel), then run at 80 V for 180 minutes (when the sample is just out of
on seperating gel).
After electrophoresis, the gel was stained with Coomassie Brilliant Blue for 15-20 min to
locate the protein bands. The gel was then washed with destaining buffer until all color was
removed [115].
2.2.12. Investigate some factors affecting recombinant laccase activity
2.2.12.1. pH effect
45
The enzyme solution was incubated with ABTS substrate mixed in a buffer range of pH 2 to
9, including buffer: glycine-HCl with pH 2-3; sodium acetate with a pH of 4-5 and potassium
phosphate with a pH of 6-9. To evaluate the pH stability, laccases were incubated with buffers
with pH varying from 2 to 9 for 1 h at 35°C. Measure the optical absorbance at 420 nm, compared
with the control [29]. 2.2.12.2. Effect of
reaction temperature The reaction mixture including
enzyme solution, ABTS substrate, suitable pH buffer was incubated at different temperatures
in the range of 20oC - 60oC to find the appropriate reaction temperature. To determine the thermal
stability of enzymes from the studied fungal strains,
Purified enzymes were incubated at different temperatures from 20oC - 60oC, after 1 hour with
the pH determined above. Measure the optical absorbance at 420 nm, compared with
evidence
[29]. 2.2.12.3. Effect of metal ions
The reaction mixture including enzyme solution, ABTS substrate, suitable pH buffer is
incubated with salts of metals: Cu2+, Ca2+, Mg2+, Co2+, Zn2+, Fe2+, Mn2+ at concentrations
1 mM with pH and temperature as determined above for 1 h. Enzyme activity was measured
spectrophotometrically at 420 nm, compared with the control [29].
2.2.13. Determine the value of Km and Vmax
Michaelis constant Km and maximum reaction rate Vmax of rFolac1 were determined
through the reaction between laccase and ABTS substrate with varying concentrations.
at the values of 1 µmole, 2 mole, 4 µmole, 6 µmole, 8 mole, 10 mole, 50 µmole.
The reaction mixture consisting of purified laccase and ABTS at different concentrations was
incubated in 100 mM sodium acetate buffer, pH 4.5, at 45ºC for 60 min. The products of the
reaction were determined spectrophotometrically at 420 nm as described in
2.2.3. The Km and Vmax values are determined by the equation

first
= first
+
first
nice
[]
y = ax + b. Where V is the number of micromoles of laccase reaction product and ABTS formed
in 1 min [S] is the concentration of ABTS (measured in mM/ml).
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3381512/).
forty six
2.2.14. Test for the ability to decolorize synthetic dyes 2.2.14.1. The
color treatment assay of the purified recombinant rFolac1 fungal
strain was used to test the color removal ability of 11 dyes at a concentration diluted 10
times the concentration of the colors applied in the process. testing the ability to remove dye
colors of natural strains of fungi. The process of removing dye colors is carried out according to
the reaction with the composition: 100 µl rFolac1, sodium acetate buffer solution (0.1 M), dye
color 0.001% (only Malachite green 0.0001%), adding Add distilled water to achieve total reaction
corresponds to 1 mL. The reaction was carried out at 55oC.
Samples were collected every three days and decolorization was assessed by
spectrophotometric measurement at peak absorbance. The maximum absorption wavelength
was determined using the scanning function of the absorption spectrometer. Decolorization ability is determine
determined as follows:
Decolorization capacity (%) = ((Initial absorbance) - (Final absorbance))/(Initial absorbance)
× 100% (formula 3) [84].
The dye color treatment ability was determined as Equation 3 every 4 hours at the
maximum wavelength corresponding to each dye color. 2.2.14.2.
Effect of intermediates on dye decolorization

of rFolac1
Ability to reduce dye colors in the presence of intermediates
Syringaldehyde, HOBT, Vanaline were made according to the following decolorization reaction:
100 µl rFolac1, intermediates (Syringaldehyde, HOBT, Vanillin) at 1 mM, sodium acetate buffer
(0.1 M), dye color 0.001% (Malachite green 0.0001%), add distilled water for a total reaction of 1
mL. The reaction was carried out at 55oC after 24 h at the maximum wavelength corresponding
to each dye color [40].
2.2.15. Statistical
processing Experimental data are the average of at least 3 replicates. Research results
were analyzed by ANOVA (Duncan's test) with p < 0.05 using SPSS program
version 16.0.
47
Chapter 3. RESEARCH RESULTS
3.1. Isolation of mold strains with LACCASE ACTIVE 3.1.1. Isolation,

purification and breeding of mold strains
A total of 16 plant humus samples including 6 samples collected in the green
park area and 10 samples collected in hillside forests were collected and diluted to
10-3 and 10-4 dilution concentrations for inoculation . spread on PDA agar plate. After
3 to 5 days of culture at 30°C, observing selected strains of white filamentous fungi
with strong growth colonies with different morphology will be transferred to petri dishes
containing separate PDA medium and continue to culture. 2 to 3 times to purify (Figure 3.1).
Figure 3.1. Colonies of 12 mold strains isolated from Thua Thien Hue (F1-
F12)
48
Based on the observation of colony morphology, mycelium and growth ability of
mycelium, 12 different mold strains (symbol F1-F12) collected in Thua Thien Hue province
were selected. domestication and breeding. These fungal strains will be used to test the
ability of laccase biosynthesis in the next experiments.
3.1.2. Screening mold strains that biosynthesize laccase In
order to screen mold strains capable of laccase biosynthesis, we conducted a
qualitative test for laccase production on BSM medium supplemented with guaiacol (0,01)
%) as a color indicator for laccase reactions. Colonies with laccase activity will produce
visible changes on the agar medium. If the colony has lignin-degrading enzyme activity,
then on BSM medium containing guaiacol, a purple-red degradation ring will appear for fungi.
The results showed that, from 12 isolates, 5 strains showed color reaction to guaiacol.
Through testing, comparing the color resolution area on the agar plate, we selected 3 strains
of F4 fungi; F5 and F8 with darker color region, excel in degrading guaiacol to continue to
study and preliminary quantify the ability to produce laccase of these 3 fungal strains (Figure
3.2).
Figure 3.2. Screening for peroxidase biosynthetic strains on supplemented selective media
guaiacol
49
3.1.3. Some morphological characteristics of colonies and spore-forming stalks of

isolated fungal strains
Fungal strains F4, F5 and F8 were grown on PDA medium after 3 to 5 days,
measured colony diameter, observed colony color with the naked eye and observed
colony morphology under optical microscope. .
The fast-growing strain F4 has colonies that are spreading, thick, about 6.5 cm in
diameter. The white mitochondria gradually change to a pink, spongy outer edge. The
bacteriophage is white in color. Microscopically, the mycelium is branched, septate.

Spores arise within the mycelium, with spores ovoid, located at the terminal ends or
interspersed between mature hyphae. They can grow as single stems or as shoots,
which separate and grow germ tubes if the spores meet favorable conditions (Figure 3.3).
Figure 3.3. Mycelial colonies of strains F4, F5, F8
The colony of strain F5 is round, about 6 cm in diameter, the surface of the
mycelium is thick, white, and the outer edge of the colony is translucent white. The
mycelium is pale yellow in color. Observation on optical microscopy, the mycelium is
segmented, segmented, and branched. Small ovoid or round spores are located in
mycelium, concentrated in clusters. Spore peduncle is hyaline, rhomboid, slightly curved,
pointed at the tip, unbranched, septate, usually 3 septa (Figure 3.3).
50
The colony of strain F8 has a diameter of about 4.5 cm after 3 days of culture, thick
filamentous, elongated, velvety surface, thin colony edge, root cover. The mycelium is white and
gradually turns green when the mycelium is old (the color of the spores), the substrate mycelium
grows deep, and is white. Microscopic examination showed that the mycelium was long, without
transverse septa, with large encapsulated spores. The spore-forming peduncle arises from
unbranched anaerobic bacteria, the apical part of the peduncle is rounded, the primary flask,
and the granule-like spores grow
round (Figure 3.3). 3.1.4. Effect of some culture conditions of mold strains on the activity of
by laccase
3.1.4.1. Investigate the ability to accumulate laccase in fermentation media
The culture medium is one of the very important factors in laccase biosynthesis. The
ability to accumulate laccase enzyme was investigated in semi-solid fermentation (addition of
10% substrate) and liquid fermentation (addition of 5% substrate). At each experimental
condition, BSM medium was supplemented with substrates derived from plant biomass, which
are straw and wood pulp.
The results of investigating the laccase accumulation ability of strain F4 in 4 fermentation
media, namely MF1, MF2, MF3 and MF4 over 18 days of culture are shown in Table 3.1.
Table 3.1. The ability to accumulate laccase of strain F4 in 4 fermentation media.

Lip Time (days)
school
3 6 9 twelfth 15 18
ferment
MF1 0.97±0.12 2.07±0.19 3.09±0.07 3.30±0.19 2.52±0.07 2.55±0.14
MF2 1.97±0.05 2.08±0.09 2.43±0.12 2.89±0.07 3.47±0.07 3.22±0.21
MF3 1.60±0.36 2.52±0.07 1.67±0.05 1.18±0.21 0.90±0.10 0.76±0.07
30.97±5.5 95.69±4.7 148.61±4.1 186.25±6.5 179.86±3.4

MF4 170±5.86
2 5 8 0 8
Through Table 3.1, it was found that the ability to accumulate laccase of strain F4 on MF4
medium was much higher than that of the other 3 media, approximately 200 times. Thus, the
conditions of liquid fermentation using straw flour are suitable for the laccase production ability
of strain F4. The ability to accumulate laccase is low in the first 6 days, increases after and reaches
51
Salivary value on 12th day of culture with enzyme activity reaching 186
U/mL. After that, the ability to accumulate enzymes is reduced.
Laccase activity also changed strongly according to the carbon composition of the culture
medium, under the same conditions of liquid fermentation, the medium using carbon source from straw
(186 U/mL) gave much higher activity than carbon source from wood pulp (3.2 U/mL). Thus, with strain
F4, the carbon source from straw is suitable for laccase biosynthesis.
Survey results on the ability to accumulate laccase of strain F5 on 4 conditions up to
yeast are MF1, MF2, MF3 and MF4 over 18 days of culture, shown in Figure 3.4.
5
Laccase
activity
mL)
(U/
first
0
0 3 6 9 twelfth 15 18
Fermentation time (days)
MF1 MF2 MF3 MF4
Figure 3.4. The ability to accumulate laccase of strain F5 in 4 fermentation environments
Based on the graph of Figure 3.5, it shows that strain F5 has a strong ability to accumulate laccase
better on MF4 than on the other 3. On all 4 fermentation media, the highest activity was obtained after
12 days of culture. The highest laccase enzyme activity reached 1.5 U/mL; 2.89 U/mL; 3.06 U/mL and
5.72 U/mL on 4 MF1 fermentation conditions, respectively; MF2; MF3 and MF4 after 12 days of culture.
Enzyme accumulation was higher in liquid fermentation than in semi-solid fermentation. In semi-solid
fermentation, the activity was only 1.06 U/mL, but in liquid fermentation, the activity increased 5 times
to 5.67 U/mL.
The results of investigating the laccase accumulation ability of strain F8 in 4 fermentation
media, namely MF1, MF2, MF3 and MF4 over 18 days of culture are shown in Figure 3.5.
52
3.5
2.5
Laccase
activity
mL)
(U/
1.5
first
0.5
0
0 3 6 9 twelfth 15 18
MF1 MF2 MF3 MF4
Figure 3.5. The ability to accumulate laccase of strain F8 in 4 fermentation media
The chart in Figure 3.6 shows that the ability to accumulate laccase of strain F8 on MF2
fermentation conditions was the lowest at 0.97 U/mL after 9 days of fermentation and did not change
much over 18 days of fermentation. The MF4 fermentation medium was also suitable for the laccase
production of strain F8. The obtained enzyme activity reached the highest value
on day 15 of fermentation with a value of 1.67 U/mL; 0.9 U/mL, 2.92 U/mL
and 3.07 U/mL on MF1, MF2, MF3 and MF4 medium, respectively. 3.1.4.2. Effect
of culture temperature of mold strains on the activity of
laccase
The results in section 3.1.4.1 showed that strain F4; F5 and F8 have higher ability to produce
extracellular laccase enzyme under liquid fermentation conditions than semi-solid fermentation. Because
so the ability to accumulate extracellular laccase was investigated over 3 temperature ranges of 25oC;
30oC and 35°C under liquid fermentation conditions.
The results shown in Figure 3.6 show that, strain F4 cultured on MF3 medium at 25°C and 30°C
gave higher laccase activity than at 35°C. At 25°C, the laccase activity peaked at 2.85 U/mL after 9
days of culture and dropped sharply to only 1 U/mL after 18 days of culture. At 30°C, enzyme activity
peaked (2.52 U/mL) earlier, only after 6 days of culture, and then gradually decreased to 0.76 U/mL.
At 35°C, the activity
reached a maximum of 1.92 U/mL after 9 days of fermentation.
53
2
Laccase
activity
mL)
(U/
first
0
3 6 9 twelfth 15 18

25°C 30°C 35°C
Figure 3.6. The ability to accumulate laccase of strain F4 using culture medium
MF3 at different temperatures.
For MF4 fermentation medium, the enzyme accumulation ability of strain F4 at 3

temperature levels was different. At 35°C, the activity peaked at 174 U/mL after only 6 days
fermentation, while at 25°C peaked (19.25 U/mL) after 9 days of fermentation, and at
30°C peaked at 186 U/mL after 12 days of fermentation and only decreased slightly to
170 U/mL by day 18 (Figure 3.7).
250
200
Laccase
activity
mL)
(U/
150
100
50
0
3 6 9 twelfth 15 18

25°C 30°C 35°C
Figure 3.7. The ability to accumulate laccase of strain F4 using MF4 culture medium
at different temperatures.
54
The strain F5 was also cultured at different temperatures on two types of media, MF3 and MF4. The
results showed that the ability to accumulate enzymes under fermentation conditions in MF3 medium was
highest at 25°C, peaking at 5.42 U/mL.
after 12 days of fermentation (Figure 3.8).
Laccase
activity
mL)
(U/
4
first
0
3 6 12 9 15 18
25°C 30°C 35°C
Figure 3.8. The ability to accumulate laccase of strain F5 using MF3 culture medium at different
temperatures.
When cultured on MF4 medium, strain F5 showed the ability to accumulate enzymes in
The temperature of 30oC was higher than that of the culture at 25°C and 35°C (Figure 3.9).
Laccase
activity
mL)
(U/
4
first
0
3 6 9 twelfth 15 18
Fermentation time (days) 25°C
30°C 35°C
Figure 3.9. The ability to accumulate laccase of strain F5 using culture medium
MF4 at different temperatures.
55
The ability to accumulate laccase at 30°C was 2 times higher when compared with the culture
conditions at 35°C. In addition, the peak of enzyme accumulation was at 35°C (2.57 U/mL) after 9 .
fermentation day earlier than 30°C (4.19 U/mL) after 12 days of fermentation.
Survey results on strain F8 with MF3 and MF4 medium showed that MF3 fermentation medium at
25°C was the best for laccase biosynthesis with the obtained enzyme activity reaching the maximum value
of 4.8 U/ mL after 9 days of fermentation. When carrying out cultures at 30°C and 35°C, the obtained
enzyme activity values were significantly lower than
with a temperature of 25°C, peaking at 2.92 U/mL and 2.62 U/mL respectively (Figure 3.10).
Laccase
activity
mL)
(U/
first
0
3 6 12 9 15 18
25°C 30°C 35°C
Figure 3.10. The ability to accumulate laccase of strain F8 at 3 temperature levels in the environment
MF3 . fermentation field
The MF4 fermentation medium showed no significant change in the ability to accumulate laccase at
3 different culture temperatures. The temperature of 25°C gave the best results with the enzyme activity
obtained after 9 days of culture reaching a value of 3.76 U/mL. At a temperature of 30°C, the ability to
accumulate enzymes increases steadily over the days and lasts up to 18
day reached 3.17 (U/mL) (Figure 3.11).
56
3
Laccase
activity
(U/
L)
first
0
3 6 9 twelfth 15 18
25°C 30°C 35°C
Figure 3.11. The ability to accumulate laccase of strain F8 at 3 temperature levels in the environment
MF4 . fermentation field
Through surveying the ability to accumulate laccase of F4, F5 and F8 on 4 types of media and 3
temperature ranges, it was found that MF4 fermentation medium and fermentation temperature of 30°C
were suitable for enzyme production of C. all 3 strains. Compare the ability to accumulate
laccase of 3 strains F4, F5 and F8 in the same fermentation medium MF4 and at a fermentation temperature
of 30°C, showed that the superior laccase enzyme production ability of the F4 fungal strain was nearly 200
times higher than that of the other two hybrid strains, Maximum activity was reached after 12 days of
fermentation (186 U/mL). Therefore, we selected the laccse-producing F4 strain under liquid fermentation
conditions, the medium supplemented with straw meal substrate to collect crude enzyme juice for
investigation of laccase properties from natural origin. And strain F4 was also selected as the raw material
to study to isolate the genome coding for laccase enzymes in this thesis.
3.1.4.3. Total protein content and enzyme purity Enzyme concentration
was prepared within the concentration range of the calibration curve. From the standard curve
equation: y = 0.0194x + 1.442, the concentration of enzymes can be inferred by the relationship between
concentration and optical density (OD). For an enzyme
preparation, in addition to determining the level of activity, it is also necessary to
its cleanliness must be assessed. The quantity that characterizes the purity of an enzyme is the specific
activity. Specific activity is the number of enzyme units/mg protein (U/mg). According to Table 3.1,
57
laccase from strain F4 has much higher purity than strain F5 and F8. Therefore, strain F4 was used
for further studies.
Table 3.2. Protein content and laccase activity of 3 strains F4, F5, F8
Strains Total Protein (µg/mL) Activity (U/mL) Specific Activity (U/mg)
F4 45.70±0.68 186.25±6.50 4.0±0.13
F5 59.00±2.0 5.72±0.07 0.1±0.003
F8 25.70±0.62 4.8±0.25 0.19±0.05
3.1.5. Investigate some properties of natural laccase such as the influence of pH, temperature,
degree, metal ions
3.1.5.1. Effect of pH
The pH of the medium greatly affects the rate of enzyme reactions. Because pH affects the
ionization state of the R radicals of amino acid radicals in the enzyme molecule, ionization of
functional groups in the active site and ionization of the substrate. To find the optimal pH range for
laccase activity from the enzyme-active strain F4 was
determined through the use of fermentation broth of strain F4 in MF4 medium and buffers with
different pH. Figure 3.13 shows that the enzyme produced by F4 has the strongest catalytic activity
in the pH range from 4 to 6, when increasing the pH from 3 to 5, the enzyme activity also increases
from 72.1% to 100%. The activity decreased slightly with increasing pH 6, while
89.5%, then decreased sharply when increasing pH to 8 to only 33.1% (Figure 3.13).
58
120
100
80
Remaining
activity
(%)
60
40
20
0
3 4 5 6 7 8
pH
Figure 3.12. Effect of pH on laccase activity of strain F4 3.1.5.2. Effect of
Temperature Temperature has an
effect on the rate of a reaction, with each enzyme working only at a suitable temperature
range. Therefore, in order to use enzymes with high efficiency, it is first necessary to study to
find the optimal temperature for enzyme activity. The results of studying the effect of temperature
on the activity of laccase from strain F4 are shown in Fig

3.14.
120
100
80
Remaining
activity
(%)
60
40
20
0
20 25 30 35 40 45 50
Temperature (°C)
Figure 3.13. Effect of temperature on laccase activity of strain F4 The optimum
temperature for laccase activity was determined by measuring enzyme activity at different
reaction temperatures from 20°C to 50°C. The results in Figure 3.14 show that, when the
temperature gradually increases from 20°C to 45°C, the laccase activity also increases from 20°C to 45°C.
59
66.4% to 100%, the most suitable temperature for laccase to work is about 45°C and decrease
when the temperature is increased to 50°C to 74.7%. At 20°C to 30°C the enzyme is still able to
active but the activity decreased, only about 70.2%. This is also one of the strengths of the F4
strain when using enzymes in environmental treatment at a lower temperature than the enzymes
studied, reducing consumable costs.
3.1.5.3. The influence of metal ions
In addition to the dependence on factors such as temperature, pH of laccase activity
also depends a lot on the presence and concentration of metal ions such as: Fe2+; Cu2+;
Ca2+; Mn2+; Mg2+; Zn2+; Co2+. Based on Figure 3.15, it shows that the laccase activity of
strain F4 is less affected by metal ions, only Fe2+ ions inhibit the enzyme's activity, the
enzyme is almost completely inactivated when adding 5 mM Fe2+ ions. into the reaction
mixture and the activity decreased by 96.7% compared to the control. Cu2+ ions ; Mg2+ and
Co2+ slightly increased laccase activity by 5%, respectively; 15%; 4% compared to the
control. Almost the remaining ions did not significantly change the laccase activity of strain
F4. This could be a good property when applying this enzyme in pollution remediation.
140
120
100
Remaining
activity
(%)
80
60
40
20
0
Control Fe²ÿ Mn²ÿ Mg²ÿ Zn²ÿ Co²ÿ
Metal ions
Figure 3.14. Effect of metal ions on F4 . laccase activity
3.1.6. Identification of
microorganisms 3.1.6.1. Total DNA
extraction For follow-up studies, the total DNA was obtained without breakage
fracture, high purity is very important.
60
Genomic DNA
Figure 3.15. Electrophoresis of the total DNA separation product of F4 . strains
M: GeneRuler 1 kb DNA Ladder (Thermo Scientific), 1:
total DNA product of strain F4
The results of electrophoresis test on 0.8% agarose gel, shown in Figure 3.15, show
that the total DNA from the F4 fungal strain obtained is not broken, compact, clean can be
used for further experiments.
3.1.6.2. Clone ITS fragment 1-4 by PCR The
ITS 1-4 sequence of F4 is amplified using primer pair ITS1 (5'
TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC 3'). PCR
product of ITS gene fragment 1-4 strain F4 was checked by electrophoresis on
0.8% agarose gel (Figure 3.16).
sixty one
Figure 3.16. PCR product ITS sequence 1-4 strain F4.
M: PreciGene 1 kb DNA Ladder (BioPioneer),
1: PCR product
This result shows that the band of PCR product is a single, sharp band with a size of
about 550 bp, consistent with theoretical calculations, showing that the desired gene
fragment may have been amplified specifically and with sufficient concentration. for sequence
determination. Based on colony characteristics and mycelium morphology along with coding
gene sequence, strain F4 has 99% similarity with Fusarium oxysporum species when compared on
Genbank.
The ITS sequence of F4 was determined to include 544 nitrogen base pairs, compared
with the 18S rRNA gene sequences of mold strains published on Genbank data banks,
showing that strain F4 has a high degree of similarity with fungal strains of the genus
Fusarium, species F. oxysporum. This strain is named F. oxysporum HUIB02, Genbank
code KX388184.
ITS1-4 gene sequence of strain F. oxysporum HUIB02 was compared with some other
fungal strains on Genbank: F. oxysporum f. sp. vasinfectum
EU849584, F. oxysporum F2: KY810793, F. oxysporum ELRF 8: KX786247, F.
oxysporum CBS 140424: KT794176, F. solani OUCMBI110106: KP269017, F.
lateritium CBPPR0050: KT211542, F.proliferatum NOB110: GU363955, F. incarnatum
HNMi: KX184815 using Mega X software (Figure 3.18).
62
Figure 3.17. Species phylogenetic tree of strain F. oxysporum HUIB02 and some
other Fusarium species on Genbank.
The tree with the highest likehood similarity is selected. The digits represent the percentage
of the phylogenetic tree that is related to the next taxonomic group.
3.2. CREATION OF LACCASE ENGLISHING GENES FROM Fusarium oxysporum
3.2.1. Amplification of laccase genes from F. oxysporum cDNA In
order to isolate the laccase genes of F. oxysporum, the exon sequences of the genes
are amplified from the F. oxysporum cDNA. F. oxysporum cells cultured on BSM
medium were collected and extracted total RNA to create raw materials for cDNA
biosynthesis. The results of total RNA extraction are shown in Figure 3.19. The total RNA
electrophoresis image showed the appearance of two clear, clean, and less broken RNA
bands corresponding to the positions of the 18s and 28s ribosomal RNA subunits. The
results show that the quality of the obtained RNA is guaranteed for cDNA biosynthesis.
Figure 3.18. Electrophoresis of total RNA products from F. oxysporum HUIB02

M: GeneRuler 1 kb DNA Ladder (Thermo
Scientific), 1: Total RNA of F. oxysporum HUIB02
From the total cDNA, the genes encoding laccase 1 (Folac1), laccase 2 (Folac2),
laccse 3 (Folac3) and laccase 4 (Folac4) from F. oxysporum were successfully amplified.
However, after nucleotide sequencing, only Folac1 gave theoretically consistent results
such as no intron region, no triplet terminator sequence. Therefore, the thesis framework
only presents the results of cloning Folac1 from cDNA. The remaining laccase-coding genes
63
(Folac2, Folac3 and Folac4) were amplified from total DNA to check the presence of
the presence of these genes in the F. oxysporum genome.
Results of amplification of Folac1, Folac2, Folac3 and Folac4 genes by special primer pairs
brand Folac1F/ Folac1R; Folac2F/ Folac2R; Folac3F/ Folac3R; Folac4F/ Folac4R
by PCR is shown in Figure 3.20.
The results showed that the Folac1 genes were successfully amplified from cDNA and
Folac2, Folac3 and Folac4 were successfully amplified from total DNA, the product
PCR on 1% agarose gel appeared clear band, without by-products, about 2 kb in size for Folac1 and
Folac3, 2.2 kb in size for Folac2 and Folac3.
Folac4, equivalent in size to the laccase genes of F. oxysporum f. sp. lycopersici 4278 was published
in NCBI. Therefore, the PCR product is of sufficient quality to generate a source
raw materials for cloning and gene sequencing.
A REMOVE OLD EASY
Figure 3.19. The PCR product amplifies the laccase-coding genes.
A. Folac1 gene PCR product, M: GeneRuler 1 kb DNA Ladder (Thermo Scientific), B. Folac2 gene
PCR product, M: 100 bp Molecular Ruler PCR product (Bio-Rad), C. Folac3 gene PCR
product, M : 100 bp Molecular Ruler PCR (Bio-Rad), D. Folac4 gene PCR products, M: 100
bp Molecular Ruler PCR products (Bio-Rad).
3.2.2. Clone genes encoding the enzyme laccase in the pGEM®T-Easy . vector
PCR products corresponding to 4 genes Folac1, Folac2, Folac3 and Folac4 were attached to
pGEM®T-Easy cloning vector and transformed into E. coli Top10. The E. coli transform carrying the
recombinant vector pGEM®T-Folac1, pGEM®T-Folac2, pGEM®T-Folac3, pGEM®T-Folac4 was
tested based on colony direct PCR and cleavage method. by restriction enzymes.
sixty four
3.2.2.1. Initial screening by PCR directly from colonies The

PCR method directly from colonies gives initial screening results (results are shown
in Figures 3.21, Figure 3.22, Figure 3.23 and Figure 3.24).
Figure 3.20. Folac1 gene PCR product from recombinant colonies
M: GeneRuler 1 kb DNA Ladder (Thermo Scientific), 1-2:
PCR product of colonies carrying Folac1 gene with specific primer pairs
Figure 3.21. Folac2 gene PCR product from recombinant colonies.
M: PreciGene 1 kb DNA Ladder (BioPioneer), a
PCR product of a colony carrying the Folac2 gene with a specific primer pair.
The results in Figure 3.21 and Figure 3.22 show that, when amplifying PCR directly
from recombinant colonies, there are specific bands with the same size as those of Folac1
and Folac2 amplified from cDNA and total DNA . are 2 kb and 2.2 kb respectively. The
PCR product was clear, without any by-products, indicating that the Folac1 and Folac2
genes were transformed into E. coli Top10 cells.
65
M: PreciGene 1 kb DNA Ladder (BioPioneer), 1: PCR
product of bacteria carrying Folac3 gene with specific primer pairs
M: GeneRuler 1 kb DNA Ladder (Thermo Scientific), 1-2:
PCR product of colonies carrying Folac4 gene with specific primer pairs.
The electrophoresis results in Figure 3.23 and Figure 3.24 also showed only a specific band
of about 2 kb and 2.2 kb in size, exactly the same as the size of the Folac3 and Folac4 genes
amplified from the total DNA of F. oxysporum. Thus, two genes Folac3 and Folac4 were also
transformed into E.coli Top10 cells.
3.2.2.2. Examination of recombinant plasmids by cleavage reaction with EcoRI
66
The recombinant colonies were multiplied by biomass in 5 mL of liquid LB medium supplemented
with the selection factor ampicillin at a concentration of 50 µg/mL and cultured overnight at 37ÿ. Then,
collect the culture, extract the recombinant plasmid from E. coli Top10 cells using the GeneJET
Plasmid Miniprep K0502 kit (Thermo scientific, USA). Examination of recombinant plasmids by
cleavage with the restriction enzyme EcoRI.
The results presented in Figure 3.25 show that the recombinant plasmid Folac1 in closed loop
is about 5 kb in size, besides that there is a band with a different size of 4 kb. This difference may be
due to the plasmid in different supercoiled states leading to different migration rates on the gel. PCR
reaction with Folac1 primer pair using recombinant plasmid DNA as template for product size is
approximately 2 kb.
In addition, the product of cutting the recombinant plasmid by EcoRI enzyme appeared in 3 bands with
different sizes, where 1 tape has a size of 3 kb corresponding to the size
plasmid pGEM®T-Easy, the second band has a size of 2 kb corresponding to the Folac1 gene and
the other band has a size of about 4 kb which is the amount of plasmid left after the restriction reaction.
Since the vector pGEM®T-Easy has 2 recognition sites of the restriction enzyme EcoRI located at the
2 ends of the insert, when using EcoRI to cut the recombinant plasmid always results in at least 2 DNA
bands of which 1 the tape is about 3 kb in size. The above results demonstrate that the Folac1 gene
has been successfully cloned into the pGEM®T-Easy vector.
sixty seven

Folac1.
M: GeneRuler 1 kb DNA Ladder (Thermo Scientific) 1:

recombinant plasmid pGEM®T-Folac1 isolated from E. coli TOP10; 2:
the Folac1 gene PCR product on the template DNA is the recombinant plasmid
pGEM®T
Folac1; 3: recombinant plasmid cleavage product pGEM®T-Folac1 by EcoRI enzyme.
Figure 3.26 shows that the recombinant Folac2 plasmid in the closed form is
about 5 kb in size, besides that there is also a band with a different size of 4 kb because
the plasmid in different super-twist states leads to the displacement rate. on different gels.
PCR amplification reaction with Folac2 primer pair using recombinant plasmid DNA as
template for DNA cassette with size approximately 2.2 kb. Recombinant vector cutting
reaction with EcoRI restriction enzyme formed 3 bands, of which 1 band was 3 kb in
size corresponding to the pGEM®T-Easy vector size. The second band is 1.5 kb in
size and the other is 0.7 kb in size, the sum of these two bands is the size of the Folac2
gene. This is explained by the fact that in the vector pGEM®T Easy there are 2
recognition sites of the restriction enzyme EcoRI located at the 2 ends of the insert,
and in the Folac2 gene sequence there is also an additional EcoRI cleavage site .
Thus, the Folac2 gene was successfully cloned into the pGEM®T-Easy vector.
68

Folac2.
M: PreciGene 1 kb DNA Ladder (BioPioneer),
1: The Folac2 gene PCR product on template DNA is the recombinant plasmid pGEM®T
Folac2;
2: Recombinant plasmid cleavage product pGEM®T-Folac2 by EcoRI enzyme; 3:
Recombinant plasmid pGEM®T-Folac2 isolated from E. coli TOP10.
As shown in Figure 3.27, the recombinant Folac3 plasmid in the ring closed form has
a main size of about 5 kb, besides that because the plasmid can exist in different super-twist
states, resulting in different translation rates on the gel. so there is also a tape with the size
of 4 kb. PCR amplification with Folac3 primer pair using recombinant plasmid DNA as
template for DNA cassette with size approximately 2 kb. When performing recombinant
plasmid cleavage with EcoRI . restriction enzyme
yielding 2 DNA bands, in which 1 band has the size of 3 kb corresponding to the size of the
plasmid pGEM®T-Easy and the other has the size of 2 kb corresponding to the Folac3 gene.
Since the vector pGEM®T-Easy has 2 recognition sites of the EcoRI restriction enzyme
located at the 2 ends of the insert, when using EcoRI to cut the recombinant plasmid always
results in at least 2 DNA bands in it. One band is about 3 kb in size and the other is the
inserted gene segment. These results demonstrated that the Folac3 gene was successfully
cloned into the pGEM®T-Easy vector.
69

Folac3.
M: PreciGene 1 kb DNA Ladder;
1: the Folac3 gene PCR product on template DNA is the recombinant plasmid pGEM®T
Folac3;
2: recombinant plasmid cleavage product pGEM®T-Folac3 by EcoRI enzyme; 3:
recombinant plasmid pGEM®T-Folac3 isolated from E. coli TOP10.
Electrophoresis of the recombinant vector pGEM®T-Folac4 (Figure 3.28) showed,
This plasmid has a main band of about 5 kb-6 kb in size, a sub-band of about 3 kb and a
band less than 0.25 kb. The main band is about the same size as the vector (3 kb) plus the
Folac4 gene, while the 3 kb band could be a recombinant plasmid that exists in supercoiled
form, the low band is total RNA. The cleavage reaction was limited by EcoRI for 2 bands
with sizes of 3 kb and 2.2 kb, corresponding to the size of the straight-chain pGEM®T vector
and the inserted Folac4 gene fragment. The PCR reaction product is a band of 2.2 kb in
size. Therefore, it can be confirmed that the Folac4 gene has been successfully cloned into
the pGEM®T-Easy vector.
70
Figure 3.27. Restriction product by EcoRI recombinant plasmid pGEM®T- Easy

Folac4
M: PreciGene 1 kb DNA Ladder (BioPioneer)
1: Folac4 gene PCR product using recombinant vector pGEM®T-Folac4 as template DNA; 2:
Recombinant
vector cutting reaction product pGEM®T-Folac4 by EcoRI enzyme; 3: Recombinant plasmid
pGEM®T- Folac4 isolated from E. coli TOP10
3.2.3. Sequencing laccase Plasmid
gene carrying recombinant vector of each gene Folac1, Folac2, Folac3 and Folac4
was submitted for nucleotide sequencing by the company FIRST BASE (Malaysia).
3.2.3.1. Folac1 . gene sequence
The Folac1 gene is cloned from cDNA with size 2064 nucleotides, so in
nucleotide sequences obtained without introns, were registered on genBank with code MT362052.
The deduced amino acid sequence obtained from the results of sequencing the recombinant
plasmid carrying the Folac1 gene was compared with the sequence obtained from
species F. oxysporum f. sp. lycopersici 4287: XM_018392545, F. odoratissimum NRRL
54006: XM_031207509, F.oxysporum NRRL 32931: XM_031188070, F.
proliferatum ET1: XM_031231991, F. fujikuroi IMI 58289: XM_023580876 from data on genBank
using Mega X software (Figure 3.29).
71
48 Fusarium oxysporum HUIB02

91
Fusarium oxysporum NRRL 32931
Fusarium oxysporum f. sp. lycopersici 4287
Fusarium odoratissimum NRRL 54006
Fusarium proliferatum ET1
100 Fusarium fujikuroi IMI 58289
Figure 3.28. Genetic genealogical tree of Folac1 gene and some other laccase genes
on Genbank.
The tree with the highest likehood similarity is selected. The digits represent the percentage of the
phylogenetic tree that is related to the next taxonomic group.
The results of using Protein Homology/analogy Recognition Engine V 2.0 software to analyze
homologous protein structure and identify functional regions showed that Folac1 has a similar structure
to laccase from Thielavia arenaria with 100% confidence . Based on the spatial structure of TaLcc1
protein in T. arenaria (code c3ppsD) the spatial structure of Folac1 was built. The results show that the
predicted secondary structure of Folac1 has 7 ÿ-coil folds and 38 ÿ-sheet folds (Figure 3.30).
The Folac2 gene has a length of 2229 nucleotides and contains 5 non-coding regions with the
lengths of regions of 52 nucleotides, 48 nucleotides, 52 nucleotides, 55 nucleotides, 47 nucleotides,
which have been registered on genBank with code KY825237.
The deduced amino acid sequence obtained from the results of sequencing the recombinant
plasmid carrying the Folac2 gene was compared with the sequence obtained from F. oxysporum species.
f. sp. lycopersici 4287: XM_018393137, F. odoratissimum NRRL 54006:
72
XM_031210768, F. oxysporum NRRL 32931: XM_031192261, F. proliferatum
ET1: XM_031228032, F. verticillioides 7600: XM_018901706, from data on genBank using Mega X
software (Figure 3.31)

70


100 Fusarium verticillioides 7600
Figure 3.30. Genetic genealogical tree of Folac2 gene and some other laccase genes
on Genbank.
The spatial structure model of Folac2 is built based on the structural model of the Cu-oxidizing
enzyme from T. arenaria. The ÿ-plate structure is shown by thin-slice arrows; The ÿ-helical coil
structure is presented as a thin slice
scroll style.
Through the Protein Homology/analogy Recognition Engine V2.0 software, Folac2 has a
structure similar to laccase enzyme from Thielavia arenaria with 100% confidence. Based on the
spatial structure of the TaLcc1 protein in T. arenaria (code c3ppsD), the spatial structure of Folac2
was built. The results show that the predicted secondary structure of Folac2 has 7 ÿ-coil folds and
36 ÿ-sheet folds (Figure 3.32).
seventy three
The purified pGEM®T-Folac3 recombinant vector was used as a template for nucleotide
sequencing of the Folac3 gene. The results showed that the Folac3 gene has a length of 1957
nucleotides equal to the size of the Folac3 gene template of strain F. oxysporum f.
sp. lycopersici 4287. The complete Folac3 gene sequence is registered on Genbank with code
KY825238. Comparison with the blast tool on Genbank showed that Folac3 is 99% similar to the
Folac3 gene of strain F. oxysporum f. sp. lycopersici 4287. The two genes have a total of 17 different
nucleotides at positions 123, 446, 453, 602, 617,
630, 806, 930, 994, 998, 1071, 1170, 1232, 1316, 1617, 1847, 1886. Folac3 gene has
two intron regions with the first intron length 47 nucleotides and the second intron 56
nucleotides.
The Folac3 gene encodes a protein with a length of 617 amino acids, 98% homology and has
The 10 amino acids differ from the Folac3 gene of strain F. oxysporum f. sp. lycopersici 4287. Folac3
peptide search analysis showed that no peptide signal exists, suggesting that Folac3 most likely
encodes the intracellular enzyme laccase of F. oxysporum. The N-type glycosylation analysis showed
that the Folac3 sequence has 7 Asn-Xaa-Ser/Thr sites that are capable of binding to radicals.
carbohydrates.
The deduced amino acid sequence obtained from the sequencing results of the recombinant
plasmid carrying the Folac3 gene was compared with the sequence obtained from F. oxysporum species.
74
NRRL 32931: XM_031192310, F. oxysporum f. sp. lycopersici 4287:
XM_18393180, F. Odoratissimum NRRL 54006: XM_031210721, F. proliferatum
ET1: XM_031228099, F. verticillioides 7600: XM_018901654 from data on genBank using
Mega X software (Figure 3.33).

92

100 Fusarium verticillioides 7600
Figure 3.32. Genetic genealogical tree of Folac3 and some other laccase genes
on Genbank.
The tree with the highest likehood similarity is selected. The digits represent the percentage
of the phylogenetic tree that is related to the next taxonomic group.
The spatial structure model of Folac3 is built based on the structural model of the Cu-
oxidizing enzyme from T. arenaria. The ÿ-plate structure is shown by thin-slice arrows; The ÿ-
helical coil structure is presented as a coiled thin slice. Using software to analyze homologous
protein structure and identify functional regions, Folac3 is structurally similar to laccase
enzyme from T. arenaria with 100% confidence. Based on the spatial structure of laccase
protein in T. arenaria (code c3ppsD), the spatial structure of Folac3 was built and presented.
shown in figure 3.34.
75
The results show that the predicted secondary structure of Folac3 has 6 folding positions
ÿ and 30 folding positions of the ÿ plate.
The Folac4 gene has a length of 2238 nucleotides and contains 5 non-coding regions with the lengths
of the regions being 53 nucleotides, 50 nucleotides, 59 nucleotides, 52
nucleotides, 47 nucleotides. Unlike prokaryotes, coding genes in eukaryotes usually consist of coding
regions (exons) and noncoding regions (introns). After DNA-to-RNA transcription, the cell removes the non-
coding region and joins the coding regions into a complete mRNA strand capable of translating into a
polypeptide chain. Nucleotide sequences have been registered on genBank with code KY825239. After
removing the non-coding regions, the Folac4 gene nucleotide sequence was translated into a sequence of
657 amino acids in length (Figure 3.35).
sixty one
34
Fusarium proliferatum HUIB02
48
52
Fusarium proliferatum NRR 3107
Fusarium verticillioides 7600
98 Fusarium tjaetaba FTJAE 11488
Fusarium subglutinans FSUBG 6092
27 Fusarium proliferatum ET1
76 Fusarium fujikuroi IMI 58289
Figure 3.34. Genealogical tree of Folac4 gene and some other laccase genes
on Genbank.
The spatial structure of proteins encoded by Folac4 was analyzed using Phyre2 protein structure and
function prediction software. The secondary structure of Folac4 contains 4 alpha helices and 37 ÿ-sheet
folding chains (Figure 3.36).
76
Figure 3.35. 3D spatial structure model of Folac4 F. oxysporum HUIB02 3.3. Expression
of the LACCASE ENcoding gene in Pichia pastoris 3.3.1. Cloning
cFolac1 into pGEM®T-Easy and transforming into E. coli Top10
We chose cFolac1 to further study the production of recombinant laccase through
the host P. pastoris. Therefore, the cFolac1 PCR product amplified using primers
expressed from cDNA was cloned back into pGEM®T-Easy and transformed into E. coli
Top10 by heat shock method. Then, the recombinant E. coli cells carrying the pGEM®T-
Easy/cFolac1 plassmid were selected by PCR.
3.3.2. Cloning into pPICZÿA expression vector
The cFolac1 gene fragment was cut from pGEM®T-Easy/cFolac1 cloning vector
by EcoRI and XbaI, and then attached to pPICZÿA vector also treated with the above 2
enzymes to form recombinant vector pPICZÿA / cFolac1. The cleavage product
electrophoresis showed that the cleavage reaction occurred completely, forming two
bands with sizes of 3 kb (pGEM®T-Easy vector) and 2 kb (cFolac1 gene), respectively .
These products will be purified using the GeneJET Gel Extraction K0691 kit (Thermo
scientific, USA), the purified products are checked by 0.8% agarose gel electrophoresis (Figure 3.37)
77
Figure 3.36. Cut reaction products using EcoRI and XbaI.
M: GeneRuler 1 kb Plus DNA Ladder (Thermo scientific), 1-2:
cleavage product pGEM®T-Easy/ cFolac1,
3-4: vector pGEM®T-Easy/ cFolac1
As Figure 3.38 shows, cFolac1 from pGEM®T-Easy/Folac1 and pPICZÿA were
completely purified and had the correct size (2 kb and 3.6 kb) and met the requirements
for the next steps.
Figure 3.37. Purified products by EcoRI and XbaI

M: GeneRuler 1 kb DNA Ladder,
1-4: purified Folac1 gene, 5: vector pPICZÿA
From the purified restriction product, the cFolac1 gene was attached to the straight-
chain pPICZÿA, and the binding reaction mixture was transformed into E. coli Top10 cells.
78
by heat shock method. Then continue to select white colonies carrying recombinant vector,
check by PCR reaction with primer cFolac1F/ cFolac1R. Randomly tested colonies all
appeared in bands about 2 kb in size, exactly the same as the cFolac1 gene. This proves
that, successfully attached and transformed the recombinant vector pPICZÿA/cFolac1 into
E. coli Top10 host cells .
After successful binding of the cFolac1 gene to pPICZÿA, the recombinant vector
pPICZÿA/cFolac1 was transformed into cells expressing P. pastoris according to the
method described above. Colonies of recombinant P. pastoris were screened with 100 µg/
mL zeocine antibiotic on YPDS solid medium. Colonies grown on YPDS medium were
biomass multiplied and total DNA extracted to check for the presence of the Folac1 gene
located in the host cell genome. Using primer pair AOX1, PCR reaction with the template
as total DNA of recombinant P. pastoris was carried out. The results showed that, when
using primer pair AOX1 appeared two bands with sizes of 2.4 kb and 2.2 kb, respectively
(Figure 3.39).
Figure 3.38. PCR product cFolac1 using primer pair AOX1.

M: GeneRuler 1 kb DNA Ladder,
1-4: cFolac1 PCR product from total DNA of recombinant P. pastoris
The 2.4 kb band is the cFolac1 gene fragment (the cFolac1 gene is 2 kb in size
plus the approximately 400 bp AOX1 fragment located in pPICZÿA). The 2.2 kb band is
the AOX1 gene fragment available in the genomic DNA of P. pastoris X33. Thus, cFolac1
was successfully inserted into the genomic DNA of P. pastoris X33 cells.
79
3.3.3. Optimization of recombinant laccase
expression 3.3.3.1. Effect of induction time
2500
2000
Laccase
activity
(U/
L)
1500
1000
500
0
first 25 3 4 6
Induction time (days)
Figure 3.39. Recombinant Folac1 activity over induction time.
Recombinant P. pastoris X33 yeast cells carrying the Folac1 gene were grown in
YP medium supplemented with 1% methanol to produce recombinant rFolac1 extracellularly.
Figure 3.40 shows that over time of culture, recombinant rFolac1 activity gradually
increased and peaked at 2302.8 U/L on day 4, laccase started to accumulate in 1%
methanol-induced medium on culture day. day 1. Laccase activity started to decrease on
day 5, however on the last day of culture, enzyme activity was still higher than on day 1 of
culture. This shows that recombinant laccase is still produced and accumulated until the
last culture day of the trial. 3.3.3.2. The effect of cell
density
In this experiment, the effect of cell density on laccase production was investigated
with OD600 from 0.5 to 2. Recombinant yeast P. pastoris X33 cells carrying Folac1 gene
were cultured in medium. YPM induction field by addition of methanol at times OD600 is
0.5; first; 1.5; 2 and maintained at a concentration of 1% methanol for 6 days for laccase
production. From the results, the OD value = 0.5; OD = 1; OD = 1.5 and OD = 2 laccase
activities both had a marked increase or decrease between
time intervals (Figure 3.41).
80
2500
2000
1500
Laccase
Active
(U/
L)
1000
500
0
first 2 3 4 5 6
OD 0.5 OD 1 OD 1.5 OD 2
Figure 3.40. Recombinant Folac1 activity according to cell density.
If the input cell density was equal to 1, the enzyme activity increased rapidly, steadily from
day 1 (2049 U/L) to day 4 (2302.8 U/L) and decreased slowly from day 4 (2302.8). U/L) to the last
day (2186 U/L). However, for OD = 2, the laccase activity was unstable, increasing slowly from
day 1 (1068 U/L) to day 2 (1152 U/L) and did not change much at day 3 (1176 U/L) /L). On the 4th
day, there was a strong increase (1335.7 U/L), from the 4th day, the enzyme activity decreased
slowly. At the OD value of 1.5, it shows that the laccase activity increases slowly and steadily from
the first day (1503.7 U/L) to
on the fourth day (1779.8 U/L) and after the fourth day, it decreased slowly. Laccase activity at the
time of OD = 1 increased gradually from day 1 (2049 U/L) to day 3 (2151.9 U/L) and the mutation
increased at day 4 (2302.8 U/L). and after the 4th day it gradually decreased.
Thus, it can be concluded that the input cell density before induction at the
OD600 value = 0.5; OD600 = 1; OD600 = 1.5 and OD600 = 2, at the OD600 = 1 value, the overall
activity of laccase enzyme reached the highest value of 2302.8 U/L. This can be explained because
the density of cells OD600 = 0.5 is the time when cells start to adapt, so the number of cells does
not increase or increases very slowly, leading to protein biosynthesis in the sensitive stage. low
response (about 778 U/L on the 4th day). Meanwhile, the OD value at time 1 is the time when the
growth rate of cells increases rapidly. So here the yeast cells enter the protein synthesis phase
leading to the laccase being released
81
magnified outward. With initial cell density OD600 = 1.5; The ratio of cells on the substrate is
reduced, so the growth and development of P. pastoris is slow, and the cell's metabolism is
mainly used for biosynthesis of recombinant enzymes. Therefore, the amount of extracellular
protein secreted almost did not change much and the laccase activity obtained did not change
much compared to the initial cell density OD600 = 1.0. When adding cell density with OD600 =
2, it is possible that the number of cells per nutrient in the medium is imbalanced, leading to
unstable development of P. pastoris , reduced growth, and poor growth. unstable protein
biosynthesis.
In most, inlet cell densities at all time points from 0.5 to 2 laccases started to release from
day 1 with 1% methanol addition and decreased activity.
after 5 days, however the 5th day activity was higher than the 1st day activity. That indicated,
recombinant laccase was produced and accumulated continuously until the last day of testing.
3.3.3.3. Effect of Inducer concentration
Study on the effect of induced methanol concentration
on the laccase expression yield of the recombinant P. pastoris X33 line, showed that
increasing the methanol concentration increased the expression yield and reached the maximum.
3122.4 U/L at 2% concentration (Figure 3.42).
3500
3000
2500
Laccase
activity
(U/
L)
2000
1500
1000
500
0
first 245 3 6
Methanol 0.5% Methanol 1% Methanol 1.5%
Methanol 2% Methanol 2.5% Methanol 3%
Figure 3.41. Recombinant Folac1 activity according to methanol inducer concentration.
82
At different methanol concentrations, laccase activity increased gradually from day .
from the 4th day and from the 4th day onwards, the enzyme activity gradually decreased.
At 2% methanol concentration, the highest laccase enzyme activity on day 4 was 3122.4 U/
L. Methanol at concentrations of 1% and 1.5%, the laccase activity was unstable from day 4.
Enzyme activity from day 4 to day 5 decreased and then increased again from day 5 to day 6.
This proves that the induction of P. pastoris X33 laccase enzyme production has taken place
successfully in the second stage. This second stage takes place after biomass multiplication, the
yeast strain needs to synthesize the necessary components for using methanol as the sole carbon
source, so from day 1 to day 4 of induction, enzyme activity increases. .
Biomass and expression decreased when methanol induction was below 2% or above 2%.
The etiology may be due to carbon deficiency when induced less than 2% or cytotoxicity when
induced methanol concentration is above 2% [85].
Although the addition of some inducers can increase the concentration of laccase or
generate new isoforms of the enzyme, methanol has also been shown to be toxic to yeast cells.
Therefore, too high concentrations of methanol and accumulation over a long period of time can
cause cell death along with ecological limits of yeast strains whose enzymes decrease after the
4th day of culture.
Overall, recombinant protein expression in P. pastoris X33 is closely related to the control
of methanol concentration. At an appropriate concentration will induce the active AOX promoter
that controls foreign gene expression. But at high concentrations, excess methanol can be toxic to
cells, reduce the activity of the AOX promoter, and can even cause cell death. Thus, when using
a source of inducer substrates with appropriate concentrations, it is possible to maximize the
biosynthesis of recombinant enzymes. 3.3.3.4. Effect of incubation temperature
Reducing the temperature below 35oC has been reported to have a positive effect on protein
production in P. pastoris X33, in which the investigated temperature range is usually from 25oC to
35oC. Therefore, we have conducted an experimental study to investigate the culture temperature.
During the culture process, foreign enzymes are acquired
83
Cells of the recombinant P. pastoris strain were performed in YPM medium. The culture temperature
was changed in the experimental samples from 25oC to 35oC.
4000
3000
Laccase
activity
(U/
L)
2000
1000
0
first 2 3 4 5 6
Induction time (day) 25ÿC 35ÿC

30ÿC
Figure 3.42. Recombinant Folac1 activity according to culture temperature.
As Figure 3.43 shows, at 25oC laccase activity was highest at day 4 (3280.2 U/L). Enzyme
activity increased slowly from day 1 (2641 U/L) to day 4 (3280.2 U/L), only on day 5 laccase activity
decreased slightly (2896.1 U/L). At 30oC, enzyme activity was highest on day 4 (3122.4 U/L). Enzyme
activity gradually increased from day 1 (2786.35 U/L) to day 4 (3122.4 U/L) and after day 4, enzyme
activity decreased. Enzyme activity on day 6 was lower than on day 1. This demonstrates that the
laccase accumulated on the last day is ineffective, and the yeast cells have reached the death phase.
At 35°C, enzyme activity was highest at day 4 (2813.8 U/L). From day 1 to day 4, enzyme activity
gradually increased, after day 4, enzyme activity decreased and it seems that laccase activity on the
first day did not change much compared to the last day.
At the induction temperature of 25°C, protein expression was better and increased gradually over
time up to day 4 and laccase enzyme activity on the last day was higher than in the first days. This is
also consistent with the recommendations of the Pichia expression kit (Invitrogen), the optimum growth
temperature of P. pastoris X33 is 30oC. However,
In this experiment, the induction at 30oC the laccase activity obtained was slightly worse
84
compared to 25°C. Laccase expression was lowest when expressed at the threshold temperature
35oC.
Therefore, we conclude, at 25oC , the recombinant yeast P. pastoris X33 biosynthesize and
secrete laccase optimally. This is probably because the higher the temperature, the faster the laccase
decreases, and the intracellular enzymes that catalyze the metabolic processes of the cell are also
affected, so the yeast strain does not carry out the biological process. can synthesize proteins. This
statement can also be explained that at the temperature from 35oC, the enzyme activity is lower
because when the temperature is high, the metabolism also increases very quickly, so many
substances (such as diacetyl) are produced. inhibited the growth of yeast, denatured the protein,
causing the yeast to die gradually. 3.3.3.5. Influence of shaking speed In addition to
nutritional factors, culture time,
concentration of inducers, etc., culture conditions are also a factor affecting the growth of yeast
as well as the ability to protein synthesis.
4000
3000
Laccase
activity
(U/
L)
2000
1000
0
first 24 3 5 6
Induction time (days) 180 rpm 210
150 rpm rpm 240 rpm
Figure 3.43. Recombinant Folac1 activity according to shaking speed.
With shaking speed of 150 rpm, 180 rpm and 240 rpm, laccase activity did not change much
between induction days. However, at shaking speed of 210 rpm, laccase activity increased gradually
from day 1 (2404 U/L) to day 3.
(3218.4 U/L), on the 4th day it increased sharply and after the 4th day, the enzyme activity decreased
85
gradually. Laccase activity at day 6 was higher than that of day 1. This proves that, laccase is still produced
to the last day.
From this result, it can be concluded that recombinant laccase was best produced at shaking speed of
210 rpm on day 4 (3552.8 U/L). This is appropriate because the growth (biomass) of yeast is aerobic
respiration and depends on the dissolved oxygen content of the culture fluid. Low shaking speed leading to
low oxygen content will limit the growth of yeast and vice versa when shaking speed is too high, the mechanical
forces are large, the oxygen content in the solution is high, making the oxidation - reduction potential. increase
inhibits the metabolism of cells, inhibits the growth of yeast biomass and thus the resulting enzyme activity will
be low.
3.3.4. Enzyme purification
Protein laccase is expressed by the pPICZÿA expression vector system, so
The resulting protein will be fused with the His-tag sequence, these His-tags have a strong affinity for Ni2+
ions on the affinity chromatography column. Thus, helping the recombinant protein to adhere to the sharp column
while proteins without His-tag or with only a few histidines were washed away by the low concentrations of
imidazole present in the wash buffer.
After collection of extracellular protein by centrifugation at 13000 rpm for 15 min at 4 °C, the total
protein containing laccase was placed on the previously prepared HisTrap TM FF column. After washing the
column to remove nonspecific binding proteins to the chromatographic column, the laccase protein was
ejected from the column with 0.5 M imidazole-containing propulsion buffer.
Collect the ejected fractions from the column and check the laccase activity of each. The fractions with the
highest laccase activity were selected for examination by polyacrylamide gel electrophoresis and activity was
checked by the method of Tian et al (2013) [48].
The molecular weight of purified rFolac1 is about 76 kDa. Including the enzyme's molecular mass
about 74 kDa and the signal peptide about 2.7 kDa (Figure 3.45). The results after purification showed that
the recombinant laccase was purified, the specific activity increased by 66,538 U/mg protein with a purification
efficiency of about 26%.
eighty six
Figure 3.44. Electrophoresis of the recombinant protein product of rFolac1.
M: PageRuler™ Prestained Protein (Thermo Scientific),
1: P. pastoris strain carrying the methanol-inducible Folac1 gene.
3.3.5. Investigation of recombinant enzyme
characteristics 3.3.5.1. Effect of pH on enzyme stability pH
has a great influence on enzyme activity. Each enzyme is highly active at a certain pH range.
It affects the enzyme reaction very clearly because pH affects the degree of substrate ionization and
enzyme stability. Therefore, in this experiment, the influence of pH stability on laccase activity was
investigated.
The pH range from 2 to 8 with a jump of 0.5 was used to investigate the effect
on rFolac1 activity. After 1 hour incubation with buffer at 40oC, the remaining activity of the solution
enzymes were identified.
The results in Figure 3.46 show that the enzyme laccase is stable at pH 4 to 7 (buffer glycine
HCl: 2-3; sodium acetate: 4-5 and potassium phosphate: 6-9), residual laccase activity ÿ 65%.
Enzymes are unstable in alkaline buffers pH > 7.5. At pH 7.5 the remaining activity was only < 50%
of the highest activity and at pH 8 the remaining activity was only < 43.2% of the highest activity. As
the pH increased from 2 to 4, the laccase enzyme activity also gradually increased. To pH
4.5, the enzyme activity increased sharply. Enzyme activity was incubated with sodium acetate
buffer pH 4.5. Thus, the enzyme laccase has high stability in acidic and neutral environments, and
low stability in alkaline environments.
eighty seven
100
80
Remaining
activity
(%)
60
40
20
0
2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8
pH range
Figure 3.45. Effect of pH on the stability of recombinant laccase.
3.3.5.2. Effect of temperature on enzyme stability
Under the influence of temperature, enzymes are denatured more or less and affect
the activity. Temperature can cause some of the hydrogen bonds in the hydrogen bond
network involved in maintaining the structure of the broken enzyme. The degree of influence
depends on the incubation temperature and some other factors
of the enzyme. In this experiment, laccase was incubated in sodium acetate buffer pH 4.5 at
different temperature levels 20oC, 25oC, 30oC, 35oC, 40oC, 45oC, 50oC, 55oC and 60oC
during 1 hour.
100
80
Remaining
activity
(%) 60
40
20
0
20 25 30 35 40 45 50 55 60
Temperature (Celsius)
Figure 3.46. Effect of temperature on the stability of recombinant laccase
88
The results in Figure 3.47 show that, after 1 hour of incubation with temperatures from 20oC to
40oC , the laccase enzyme activity gradually increased from 54% to 65.65%. Enzyme activity increased
sharply from 40oC to 60oC.
According to the results of this study, the enzyme only maintained 60% of its activity when treated
at temperatures below 50oC. From 55oC to 60oC , enzyme activity decreased slowly and laccase was
stable over a wide temperature range. This result can be explained because the reaction temperature
strongly affects the catalytic activity of the enzyme, each enzyme has an appropriate reaction temperature
range. In the limit of temperature that did not denature the enzyme, the enzyme activity increased
when the temperature increases. However, when the temperature is raised above the limit, the enzyme
activity decreases. The reason may be that when the temperature is high, it has broken some weak bonds
in the enzyme protein molecule, changing the structure of this molecule, especially the structure of the
active center of the enzyme, thereby affecting the structure of the enzyme. affect the catalytic activity of
enzyme.
3.3.5.3. Effect of metal ions on enzyme stability The presence of metal
ions affects enzymes in a variety of ways, which can increase or decrease the enzyme's catalytic
ability. Metal ions often affect enzyme activity in general, if not involved in the formation of the enzyme's
structure, it affects enzyme activity to the extent that it maintains the spatial configuration or increases the
rate of the reaction. either by complexing with the substrate or by participating in the binding of the
substrate to the active site of the enzyme.
In addition, metal ions play an important role in shaping the spatial structure of the enzyme molecule
or in some cases it is essential.
in the active site of the enzyme. Therefore, metal ions have a great influence on the
enzyme activity. Therefore, determining the influence of metal ions is one of the bases to guide the
production of enzymes applied in different fields.
Investigating the influence of metal ions on laccase activity showed that Mn2+ had a slight inhibitory
effect on the activity of this enzyme, the remaining activity after treatment was less than 50%. Cu2+
increased recombinant laccase activity while some other metal ions such as Zn2+, Co2+ partially inhibited
enzyme activity.
89
120
100
80
Remaining
capital
active
(%)
60
40
20
0
Ca²ÿ Mg²ÿ Mn²ÿ Zn²ÿ Fe²ÿ Cu²ÿ Co²ÿ
Metal ions
Figure 3.47. Effect of metal ions on the stability of recombinant laccase.
Enzyme activity was enhanced in the presence of Cu2+ at 1 mM, Ca2+ at 2 mM,
Zn2+ at 1 mM and Co2+ at 1 mM. In particular, Cu2+ ions strongly enhance enzyme
activity, increasing relative activity up to 132%. Thus, Cu2+ can act as a cofactor of the
enzyme, participating in the catalytic activity at the active site of the enzyme. At the same
time, unlike other enzymes, laccase has a catalytic central structure with 4 Cu atoms.
However, the laccase activity was completely inhibited by the addition of Fe2+ ions. It
shows that Fe2+ ion has the ability to not bind to the enzyme active site. This can be
explained by the interaction of Fe2+ ion with the S - S or -SH group of laccase to form a
laccase - Fe complex, which changes the conformation and reduces the enzyme activity.
The presence of Mn2+, Ca2+ and Mg2+ had a relative influence on enzyme activities below
80%. These allow for the inference that they are most likely relatively weakly linked
with the active center laccase. In addition, laccase activity is stimulated by Zn2+ ions,
possibly because mixing of Zn2+ ions is required for catalytic sites and protein structure.
Accordingly, the presence of Zn2+ ions changes the secondary structure of the protein
and increase enzyme activity.
3.3.6. Kinetics of recombinant laccase
90
The Km constant of the enzymatic reaction was determined by the ABTS concentration
range from 1 to 50 µmole. Based on the products formed of the reaction between enzyme and
substrate at different concentrations, we built a graph of the dependence of the reaction rate on
the ABTS substrate concentration by the method of Linewever-Burk (Figure 3.13). ) and the
kinetic parameters Km, Vmax are determined. The results showed that the equation of
dependence between the reaction rate and the substrate concentration obeyed the function y =
0.2268x + 0.0167 with a confidence level of 99.36% (R2 = 0.9936) .
Figure 3.48. Variation of reaction rate according to substrate concentration ABTS
The smaller the Km value, the greater the affinity of the enzyme with the substrate and vice versa.
In our study, when using ABTS as a substrate, the values of Km and
Vmax in this experiment was calculated to be 13.58 mole and 59.88 respectively
µmoles/min.
3.4. TESTING ABOUT RECOMMENDED LACCASE INDUSTRIAL Dyestuffs
Table 3.3. Synthetic dye decolorization rate of recombinant laccase
Dye decolorization efficiency (%)
Synthetic dyes Decomposition time (hours)

4 8 twelfth 24
Bromothymol blue 39.2b 41.5b 43.3b 50.0a
91
Methylene blue 24.7c 26.9bc 29.9b 31.2a
Methyl orange 43.5b 45.4b 45.1b 50.9a
Crystal violet 30.4b 36.0b 41.2b 48.2a
Remazol brilliant blue CHEAP 65.1c 76.5bc 86.0ab 88.4a
Anime blue 27.1c 45.5b 56.0a 57.9a
Evans blue 27.7b 27.9b 30.0b 46.1a
Reactive black 34.2b 52.4a 56.1a 58.5a
Indigo carmine 29.5c 40.0c 77.9b 82.7a
Orange II 13.7d 17.5c 20.4a 27.3a
Malachite green 34.5b 43.5b 54.0a 61.4a
Data are expressed as the mean of three replicates and analyzed by one-way ANOVA with p
< 0.05. The letters a, b, c, d in each row indicate a significant difference. rFolac1 is applied to
decolorize
various dyes including basic dyes (methylene blue), acid dyes (Methyl orange, Aniline blue),
reactive dyes (Evans blue), azoic dyes (Malachite). green) (table 3.2). The results showed that in
most cases, the enzyme was able to decolorize about 50% of the dye after 24 hours of reaction.
Especially rFolac1 can decolorize
82.7% and 88.4% in the case of Indigo carmine and Remazol brilliant blue R. When compared with
the decolorization activity of F. oxysporum HUIB02, recombinant rFolac1 showed a significant
improvement of 38.87%, 13.93%, 40.44% and 17.54% in the color case
Crystal violet, Aniline blue, Evans blue and Orange II.
To study the effect of mediators on drug decolorization efficiency
staining, we added syringaldehyde (1 mM), HOBT (1 mM), vanaline (1 mM) as intermediates for the
reaction and compared with control samples without the addition of intermediates (table 3.4 ) .
92
Table 3.4. The role of intermediates on dye color processing ability of recombinant laccase
Dye degradation efficiency
(%)
Decomposition time (hours)

Synthetic dyes
No substance
Syringaldehyde HOBT Vanaline
intermediary
Bromothymol blue 50.0b 59.9a 45.7b 48.7b
Methylene blue 31.2c 40.6a 37.5ab 44.8a
Methyl orange 50.9a 46.3b 45.5bc 43.0c
Crystal violet 48.2a 42.6b 46.3a 47.7a
Remazol brilliant blue CHEAP 88.4a 67.0ab 71.3a 70.6a
Anime blue 57.9b 72.2a 68.4ab 71.6a
Evans blue 46.1b 46.5b 46.3b 56.1a
Reactive black 58.5b 78.0a 55.6b 60.4b
Indigo carmine 82.7a 78.1ab 69.4b 72.4b
Orange II 27.3ab 24.6bc 30.3a 21.3c
Malachite green 61.4a 54.8ab 43.7c 46.3bc
Data are expressed as the mean of three replicates and analyzed by one-way ANOVA with p < 0.05.
The letters a, b, c, d in each row indicate a significant difference.
The results show that, with different (binding) intermediates, the decolorization efficiency will be
different (table 3.4) and there is no common intermediate for all colors. Specifically, syringaldehyde
significantly improved the activity of rFolac1 for decolorization
Bromothymol blue (55.73%) and Reactive black (75.43%); while HOBT significantly improved the
activity of rFolac1 to decolorize Orange II (34.01%); and with the substrate
Vanaline, rFolac1 degraded Methylene blue (40.22%), (46.88%) and Evans blue
(89.50%).
93
Figure 3.49. Degradability of some synthetic dyes by rFolac1.

Aniline blue (1); Evans blue (2); Reactive black (3); Indigo carmine (4); Orange
II (5); Malachite green (6); Bromthimol blue (7); Methylene blue (8); Methylene
orange (9); Crystal violet (10); Remazol blue (11).
ninety four
Chapter 4. DISCUSSION
4.1. Isolation of LACCASE . ACTIVE MOLD STRATEGIES
Laccase can be obtained from different sources such as molds, plants, bacteria, insects but
most commonly molds. Currently, many strains of filamentous fungi have been discovered that
show very good laccase synthesis such as: T. versicolor Melanocarpus albomyces [56]. In
addition, mold has the ability to grow strongly, so it is very convenient for large-scale production
of laccase for industry and life.
Wang et al (2018) also isolated Trametes sp. MA-X01 from withered plant parts, this fungus
grows well in PDA medium, with white, dense and dense flat colonies. Laccase is produced during
the growth of mycelium [95].
In 2016, the fungal strain Polyporus sp. FBD154 isolated from Bidoup - Nui Ba National
Park, Lam Dong has the ability to biosynthesize laccase with high activity of 74,925 U/L on
modified TSH1 medium with pH = 4 containing 200 g/L potato extract, first
g/L casein, 1 g/L rice bran, 5 g/L soybean meal, 10 g/L mannose, 3 g/L NH4Cl , 0.3
mM CuSO4) [2].
From 12 mold strains isolated from plant humus, we selected 3
strains F4, F5 and F8 have the ability to degrade lignin, of which strain F4 has the strongest ability
to biosynthesize laccase, reaching 186 U/mL after 12 days of fermentation. Compare with results
The results have been studied at home and abroad, strain F. oxysporum HUIB02 has very high
laccase activity, which can create many potential applications in environmental treatment,
especially in synthetic dyes. The results of screening studies for laccase activity of fungal strains
have great significance in providing an abundant source of raw materials for laccase extraction
and application.
4.1.1. Culture medium Microbial
culture medium plays an important role for microbial strains to biosynthesize different
enzymes, especially fungal strains. According to
In the study of Praveen et al (2011), high laccase activity was independent of biological
95
the mass of the fungal strain, but the laccase product has been shown to be closely related to
the culture conditions and environment, high biomass does not necessarily give laccase activity.
high [94].
Studies to enhance laccase production will contribute to the production of enzymes
needed for industrial applications as well as lignin degradation. As a secondary metabolite,
laccase production is highly dependent on culture conditions.
Laccase activity can be influenced by many factors, such as induction, pH, aeration and various
nutritional effects [21]. The potential application of laccase in Biotechnology has prompted more
research to produce industrial enzymes at commercial prices. The use of lignocellulosic waste
in laccase production reduces production costs and is also a promising method for converting
agro-industrial by-products into high-value products [98].
In recent years, various lignocellulose agricultural residues, such as rice bran, wheat
bran and barley bran have been proposed as alternative carbon sources for production of
ligninolytic enzymes by white rot fungi [8],[9]. 15]. The combination of xylidine supplementation
and glucose inhibition with lignin induction enhances activity
laccase of T. versolor to 1.58 U/mL [125].
Producing laccase with the carbon source of rice straw instead of using glucose is of
great commercial significance. According to Chunyan Xu et al (2015), the laccase activity of
T.versicolor BBEL0970 was enhanced when using straw as a substrate source, reaching 1030
U/L, an increase of 58.5% compared with the medium supplemented with glucose 5.2. U/L,
after 20 days of fermentation, showed a high potential for laccase production from rice straw
[126]. In addition to serving as a carbon source for fungal growth, lignocellulose can also act
as an inducer of laccase production and activity stabilization [113].
4.1.2. Investigation of some characteristics of natural
laccase 4.1.2.1. Effect of pH
Normally, every enzyme has a pH range in which to function, because enzymes consist
of several groups that are acidic or alkaline in nature. These groups have different ionization
conditions at different pH values [121]. Most fungal laccases are active at acidic or neutral pH
values but lose activity under alkaline conditions [68].
96
[138]. As a rule, many fungal laccases have an optimal pH in the acidic pH range that
varies depending on the type of substrate: for ABTS from pH 2.0 -5.0, for 2.6-
dimethoxyphenol in the pH range 3-8 and syringaldazine in the pH range 3.5-7.0 [85].
The optimum pH of the laccase from T. versolor 50001 is pH 2.0 [136]. Laccase from
Cryptococcus albidus exhibited maximum activity at pH 2.5 at 20oC -30oC when ABTS
was used as substrate [105]. Laccase from F. oxysporum HUIB02 also works optimally
at pH 5, similar to laccases in other strains of many studies around the world. 4.1.2.2.
Effect of Temperature
Temperature has an effect on the rate of a reaction, with each enzyme working
only at a suitable temperature range. Therefore, in order to use enzymes with high
efficiency, first of all, it is necessary to study to find the optimal temperature for enzyme
activity [87]. Some laccases have optimal activity below 35ÿC , such as laccases from G.
lucidum , which perform best at 25ÿC [58]. Laccase from T. versicolor also performed
best at 35°C [136]. Different laccases from different strains also have variations in the
optimum temperature for their activity, such as the laccase from Tricholoma matsutake
which performs best at 60ÿC [69]. Farnet et al (2008) investigated a laccase from
Marasmius quercophilus, a white rot fungus, with an optimum temperature at 80ÿC [26].
In our study, the optimal temperature for laccase activity from F. oxysporum
HUIB02 was 45ÿC, which is also within the general optimum temperature range of
laccases of different origin. 4.1.2.3. Effect of metal
ions
Although laccase is effective on a wide range of substrates, in most cases

the addition of Cu2+, Cd2+, Ni2+, Mo2+ and Mn2+ ions increases the activity.
of laccase, while Ag+ , Hg2+, Pb2+, Zn2+, Na+ and H2O2 inhibit the activity of
them [80]. Murugesan et al. (2009), studied the effect of different metal ions on laccase
activity under in vitro conditions, using crude laccase obtained from the fungus G.
lucidum. The results showed that laccase activity was enhanced by metal ions such as
Ca2+, Co2+, Cu2+ and Zn2+ at low concentrations of 1 mM. Increasing the concentration
of metal ions except Cu2+ and Zn2+ up to 5 mM or more reduces
97
enzyme activity. Among the heavy metals, Fe2+ strongly inhibits the activity of
enzyme laccase [83].
However, the use of metal concentrations at different levels affecting laccase activity
in other microorganisms has demonstrated that, at a given concentration, the activity is
maximal and then active. This feature is greatly reduced and in some cases completely
canceled. In Grammothele subargentea, it was found that at concentrations of 0.6 -1.2
mM CuSO4, laccase activity was maximal, while at higher concentrations of 1.5-1.8 mM
laccase activity decreased. 76]. A similar case was observed in T. versolor, where the
laccase activity in the presence of CuSO4 increased up to 17%; however, it decreased
significantly with high Cu2+ concentrations (80 mM) [56]. For T. hirsuta, the highest
laccase activity was 31,786 U/L when supplemented with a concentration of 5 mM [100].
According to Hernández et al (2018), when adding CuSO4, the laccase activity in the
extracellular part of wild-type and mutant strains decreased, and the laccase activity in
the intracellular part of mutant strains also decreased. that CuSO4 at a concentration of
25 µM inhibited laccase activity in F. oxysporum. f. sp.
lycopersici [34].
The extracellular and intracellular results showed that the presence of FeSO4 had
no obvious effect on laccase enzyme activity in strain F. oxysporum f. sp. lycopersici
relative to its baseline condition. This is explained by the authors, possibly because
FeSO4 does not act as an electrical conductor
death of extracellular laccase (Lac4, Lac5 and Lac9) or intracellular laccase (Lac1 and
Lac2), or the concentration used (25 M) is not the concentration required to induce induction
stimulation of laccase [34]. Robles et al (2002), tested the effect of FeSO4 at a
concentration of 5 mM on laccase activity at Chalara (syn Thielaviopsis) paradoxa CH32,
the results showed that at this concentration laccase activity was inhibited [99]. .
The laccase activity from F. oxysporum HUIB02 was also inhibited by Fe2+ and
Cu2+ increased enzyme activity. This result is also similar to laccase from some species
belonging to the genus Fusairum such as F. solani [124].
98
4.2. Isolation of LACCASE-ENCING GENES In this
study, we have successfully amplified 4 laccase-coding genes from cDNA and from
the total DNA of F. oxysporum HUIB02, including:
genes encoding laccase 1 (Folac1), laccase 2 (Folac2), laccase 3 (Folac3) and laccase 4
(Folac4). The genes Folac1, Folac2, Folac3, Folac4 have sizes of 2064 bp, respectively.
2229 bp, 1957 bp and 2238 bp. However, after nucleotide sequencing, only
Folac1 gives theoretically consistent results as no intron region, no terminator triplet
sequence. The results of nucleotide sequencing of Folac2, Folac3, Folac4 genes showed
that in the sequence of these genes, there are intron sequences and mutations appear in
the triple-terminal sequence. The occurrence of errors in the PCR reaction indicates that the
laccase genes of F. oxysporum are difficult to amplify. The cause may be due to the large
size of the gene, so the reverse transcription of mRNA into cDNA is faulty, or the error may
occur during gene amplification by PCR reaction. Thus, out of 4 isolated laccase genes, we
only obtained one complete Folac1 gene used for expression in yeast P. pastoris X33. To
investigate the functional
role of laccase isolated from fungi, some laccase genes were cloned and characterized
such as laccase from T. versicolorc [38], from Aeromonas
hydrophila [45] and from Melanocarpus albomyces [55].
Hoshida et al. (2001) used degradation primers designed based on conservative Cu-
binding regions I and IV to amplify laccase nucleotide sequences from the total DNA of T.
sanguinea M85-2 . The nucleotide sequences of the 21 clones were classified into five
groups (from lac1 to lac5) and the derived amino acid sequences were all homologous to
the known laccase sequences [37].
Cordoba Cañero (2008), isolated and identified the roles of 6 laccase genes from F.
oxysporum f. sp. lycopersici causing disease in tomato. The lac1, lac2, lac3, lac4, lac5 and
lac9 genes all have conserved regions specific for Cu binding such as those of the enzyme
phenol oxidase [16]. As of 2015, 15 laccase genes derived from F. oxysporum f. sp.
lycopersici 4287 were discovered, cloned and studied for the mechanisms involved in the
plant pathogenicity of this fungal strain [97]. Length
99
of potential F. oxysporum laccase genes ranging from 1957 to 2471 nucleotides, in which
the GC content of the gene ranges from 47% to 51% [64].
Jing Wu et al (2010), cloned the laccase gene from Aeromonas hydrophila WL 11,
nucleotide sequence analysis revealed an open reading frame of 1605 bp encoding a
polypeptide consisting of 534 amino acids [46].
To isolate the cDNA coding for laccase from Cyathus bulleri, Garg et al (2012) used
RACE PCR to obtain a laccase coding sequence of 1542 bp in length. This is a very useful
method for the isolation of gene sequences using primers from known genes and conserved
regions. The primer designed in this study was based on the analyzed 435 bp sequence,
and the primer walking technique was used to obtain the complete laccase coding sequence
[28].
Ravikumar et al (2013) cloned the 1.6 kb laccase gene isolated from Hypsizygus ulmarius
into the pET-29a (+) vector [96].
The cDNA sequence of the pclac2 laccase from the full-length Phytophthora capsici
is 1683 bp encoding a protein containing 560 amino acids, preceded by a signal peptide
with 23 amino acids. The protein sequence of pclac2 shows high similarity to other known
fungal laccases and contains four Cu atoms as the typical protein laccase.
figure [27].
In 2017, Orkun Pinar et al., expressed and characterized laccase
Cplcc1 has high redox potential from Coriolopsis polyzona MUCL 38443
The size of the Cplcc1 gene is 2106 bp and it contains 10 introns and 5 N-glycosylation
sites [92]. Thus, when compared with other research results, the laccase-coding genes
from F. oxysporum HUIB02 are highly homologous and have the characteristics of a typical
laccase.
4.3. Expression of the FOLAC1 gene that encodes EMZYME in Yeast
Pichia pastoris
4.3.1. Folac1 Gene Expression
Natural cells often produce low laccase yields that do not meet commercial needs.
Therefore, to improve production, cloning of laccase genes and their expression in other
hosts is used. Recent advances
100
Here in the field of genetic engineering has allowed the development of efficient expression
vectors for potential laccase production.
Several laccase genes have been cloned from different fungal sources such as
Phytophthora capsici and a variety of recombinant proteins have been successfully produced
when expressed in other hosts to utilize the laccase more efficiently in
biotechnology [27].
Host systems for recombinant laccase expression have also been investigated for
efficient laccase production. The P. pastoris expression system has been widely used to
produce laccases from Botrytis aclada, Ganoderma sp. En3, Fome
lignosus, Pleurotus sajor-caju, Pycnopus sanguineus, Trametes sp. AH28-2, Trametes sp.
420, T. trogii and T. Versicolor [27], [65], [107], [135] showed the suitability of this system for
laccase expression. P. pastoris secretes extracellular foreign proteins in the presence of low
levels of native proteins, especially proteinases,
thus simplifying the purification process.
Soden et al. (2002), which expressed the lac4 Psc gene from Pleurotus sajor caju in P.
pastoris, under the control of the methanol-induced AOX1 promoter, the purified laccase was
estimated to have a molecular weight of about 59 kDa [107] ].
In another study, the cDNA encoding the laccase from T. versolor was amplified by
RNA-PCR, cloned into the vectors pMETA and pMETalphaA and expressed via P. methanolica,
the molar mass of the complete laccase was 64.0 kDa [32]. Laccase isolated from the white
rot fungus Polyporus grammocephalus TR16 expressed in P. pastoris had an estimated
molecular weight of 65.4 kDa by
SDS-PAGE electrophoresis [39].
A gene encoding the laccase of T. verscolor , lccA, has been cloned and expressed in
P. pastoris; lccA gene consists of 1560 bp open reading frame encoding 519 amino acids,
the recombinant laccase was purified by ultrafiltration and precipitation (NH4)2SO4 showing
a single band on SDS-PAGE with molecular weight 58 kDa [sixty seven].
Laccase exists in many structural forms; most of them are monomers, but some are
also present in heterodimer and polymorphism. Their molecular mass ranges from 50 kDa to
140 kDa, depending on the organism, for a typical fungal laccase.
101
will range from 60 kDa to 70 kDa with an isoelectric point around pH 4.0 [85], [97].
Thus, the gene Folac1 expressed in P. pastoris isolated from F. oxysporum HUIB02 also
falls under this rule.
4.3.2. Investigation of recombinant enzyme
properties In contrast to the low yield and high cost production of laccases from
natural sources, expression of laccases through other hosts is much more suitable to meet
industrial needs. increasing. Therefore, it is very important to study and investigate the
properties of recombinant laccases, the aim is to find the optimal parameters for application.
used in industrial processes.
Some studies by other authors also gave similar results, specifically Soden et al
(2002), indicating that the recombinant laccase only increased rapidly after the 3rd day and
reached its maximum on the 5th day (about 100 U/mg) [107]. Compared with those enzymes,
the recombinant laccase in our study showed a rapid increase in activity from day 1 and
reached its maximum earlier. However, the recombinant enzyme Lacc6 in P. pastoris was
observed to reach its maximum level after 12 h of induction with 1% methanol and then the
enzyme activity decreased rapidly due to methanol toxicity [90]. This difference can be
explained by the difference in optimization for laccase production [108]. When expressing
the lac4 Psc gene from Pleurotus sajor caju in P. pastoris, with ABTS as the substrate, the
enzyme displayed optimal activity at 35ÿC and pH 3.5; The enzyme is strongly inhibited
by sodium azide and thioglycolic acid [107]. The laccase 5 coding gene TVLCC5 from T.
versicolor was overexpressed in yeast Arxula adeninivorans using the potent promoter
TEF1. The recombinant TVLCC5 protein was purified by ion affinity chromatography. The
recombinant enzyme was most active at 50ÿC at pH 4.5 [70].
Laccases from C. lagenarium expressed in P. pastoris exhibited maximum activity at
pH 3.6 - 4.0 but were more stable in buffer solutions with higher pH (> pH 3.6). The optimum
reaction temperature of the enzyme is 40ÿC, beyond which the activity decreases significantly
[12]. For the laccase gene Cplcc1 amplified from the cDNA of Coriolopsis polyzona MUCL
38443 expressed in P. pastoris was optimally active at pH 3.0, temperature 70oC [92]. The
optimal pH and temperature for laccase from T. versicolor expressed in P. pastoris are pH
2.0 and 50ÿC with ABTS substrate, the recombinant laccase is stable over the pH range from
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2.0-7.0 [67]. rLac from Cyathus bulleri laccase in P. pastoris using ABTS as substrate
had optimum pH and temperature of 4.0 and 55°C respectively, rLac stability increased
from pH 2 to pH 7 [4]. In addition, the optimal pH and temperature for laccase from F.
oxysporum expressed in P. pastoris were 6.5 and 30oC, recombinant laccase
(rFoLacc5) had the highest activity at pH 4.5 and temperature 40oC. [40]. In our study,
rFolac1 expressed in P. pastoris had an optimal operating temperature of 55ÿC at pH

5.5. Some previous publications also showed that laccase is stable in acidic
environment. According to research by Bo Wang et al. (2018), laccase from Laccaria
bicolor on yeast P. pastoris with optimum pH is 4.5 [12] or higher as the work of Li et
al (2014) indicates that pH 5.5 is the best [67]. Or the research results of Liu et al
(2003) show that pH 8 is optimal [72].
4.3.3. Optimization of recombinant laccase
expression The pH range for recombinant laccases is between 3.0 and 6.0 and
the optimum pH is 4.5. The ability of this laccase to act in weak acid buffers allows
this enzyme to be widely used. Especially, it supports the hydrolysis of lignin of
lignocellulose materials or the decomposition of synthetic dyes in acidic polluted
environments. Our results for optimal pH are consistent with the optimal pH of most previous stud
Previous research results suggest that laccase activity is optimal in weak acid buffer
solutions such as pH 3.0 (Lac2); pH 3.0 (Lacc6); pH 3.5 (Lacc12) [90], [108].
In addition, Lac1 of P. sajor- caju , after being expressed in P. pastoris and grown in
pH controlled fermentation, showed high activity in weak alkaline buffer, reaching a
maximum of 15 0.8 U/L at pH 6.0 and enzyme activity was reduced when the pH of
the buffer was acidic (only 5.7 U/L at pH 4.5) [107]. This is explained by the difference
between the optimum pH which is specific to each enzyme and the optimal pH which
also depends on the properties of the substrate related to the oxidation mechanism.
Different substrates have different oxidation mechanisms that require their own
particular pH [108], [129]. In addition, the recombinant Lac4 enzyme of P. pastoris ,
isolated from Pleurotus sajor-caju, showed different optimal pH values of 3.3; 6; 6.5; 7
corresponding to the substrates ABTS, 2,6-dimethoxyphenol, syringaldazine and
guaiacol [107]. In this study, the relative enzymatic activity of rFolac1 was maintained at
103
levels are high under acidic conditions and enzyme stability decreases if the pH of the medium
is changed to alkaline, which is different from most of the currently known laccases. In most
cases, laccase activity is more stable in the pH range 6.0-7.0, and enzyme activity is reduced
under acidic conditions [85].
Each microbial species will have a suitable pH range for different laccase biosynthesis.
Depending on species and strains, the appropriate pH value for laccase biosynthesis is acid,
neutral or alkaline. Normally, every enzyme has a pH range in which to function, because
enzymes consist of several groups that are acidic or alkaline in nature. These groups have
different ionization conditions at different pH values [117]. Most fungal laccases are active at
acidic or neutral pH values but lose activity under alkaline conditions [132]. Therefore, pH control
is necessary during culture, as some acidic proteases can be activated at lower pH and cause
adverse effects on laccase production [107].
Temperature is considered to be one of the most important factors affecting recombinant
laccase expression levels. There have been many different published works on the effect of
temperature on the biosynthesis of laccase of pastoris . Optimum temperature was yeast P.
investigated in the range of 20oC - 60oC at an increase of 10oC.
Heat stability tests were performed by incubating the enzyme in 0.1 M sodium acetate buffer
with pH 4.5 for 30 min. The results showed that the laccase activity continued to increase after
the temperature increased from 20oC to 55oC and the optimal temperature was 55oC.
After that point, the enzyme activity decreases if the temperature is still raised. The results
indicated that the optimum temperature of the recombinant laccase in P. pastoris was higher
than that of the original laccase in strain F. oxysporum HUIB02 (45oC). Some other results
showed that the recombinant laccase CcLCC6 in P. pastoris isolated from Prinopsis cinerea had
the optimum temperature at 40oC, while X33-Lac6 was active at 45oC , and Lac4 and Lac12
had the maximum temperature at 45oC . optimum at 50oC [30], [107]. Meanwhile, Hong et al
(2002) announced that it is 20oC [35]. Hong et al (2007) found that the expression activity of the
recombinant laccase increased fourfold when the temperature was reduced from 30ÿC to 28ÿC
[36]. Similar results were reported when the laccase gene was expressed in Kluyveromyces
lactis [25]. When producing the recombinant TVLCC5 laccase, culture at 20°C showed the highest biomass a
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Maximum laccase (0.35 U/mL) was also detected at this temperature, TVLCC5 secretion was
slightly lower (0.33 U/mL) at 30°C, cultured at 10°C strongly inhibited growth induces low
accumulation of the TVLCC5 laccase [70]. However, the expression of laccase from P.
sanguineus in P. pastoris was not enhanced by lowering the culture temperature, the laccase
activity in the shake culture at 30ÿC was much higher than that of 20ÿC; this could be explained
by the low growth rate of P. pastoris when cultured at 20ÿC [65].
From the above comparisons, it can be seen that rFolac1 has excellent ability to work at
high temperature, the enzyme has maintained almost 95% activity up to 60oC. This enzyme
has high thermal stability and has great potential for industrial applications.
Some publications in the world showed that OD600 = 2 is the appropriate density to
induce recombinant enzyme expression in yeast P. pastoris. According to Tang et al. (2012),
the appropriate density to induce chitinase biosynthesis in P. pastoris at OD600 = 2 [111].
However, in some other cases, the appropriate cell density may be higher, as in the case of
Plantz et al. (2006) who chose the time when the cell density reached OD600 = 5 to induce
biosynthesis . ovine interferon–ÿ recombinant (rOvINF–ÿ) [93]. Adi et al. (2012), selected the
time when the cell density reached OD600 = 2 – 6 to express human erythropoietin in P.
pastoris [5]. According to the results of this study, the best effect was achieved at OD600 = 1.
The difference between our study results and some other works may be due to the difference
between the yeast host strains P. pastoris and the conditions in proliferation, induction as well
as structure. , different sizes of protein biosynthetic gene segments.
An increase in methanol concentration caused a negative effect on laccase production.
The lower laccase activity obtained at high methanol concentrations could be due to the
accumulation of toxic products of methanol metabolism, growth retardation and reduced
biomass yield of P. pastoris [52 ] . . A concentration of 0.5% methanol produced the highest
activity of laccase from P. sanguineus in P. pastoris [65]. The highest laccase activity from B.
licheniformis in P. pastoris reached 313 ± 11 U/L, using 0.5% methanol for induction and no
cell growth inhibition was observed when methanol concentration increased to 1.5% [120].
This experiment used
105
methanol concentration from 0.5% - 3% was added to YP medium with an induction time
of 6 days. In this study, the methanol concentration required to induce laccase expression
in P. pastoris was higher than in other publications. Mei Guo et al (2008) when studying
the effect of methanol concentration on the laccase expression yield of P. pastoris strain ,
0.8% methanol was the most suitable [32]. Lu et al (2009), when studying laccase
expression in P. pastoris , determined the appropriate methanol concentration was 0.5%
[65]. Or according to Park et al. (2015) expression of laccase from P. ostreatus in P.
pastoris requires 1% methanol [90]. This difference may depend on the response time to
methanol, the physiological conditions of each strain and the ability of each strain to
respond to methanol concentrations so that when the strain uses methanol as the sole
carbon source, copper When the AOX1 promoter is activated, the target gene is expressed and secre
the target protein out.

4.4. TESTING ABOUT RECOMMENDED LACCASE INDUSTRIAL Dyestuffs
The recombinant folac1 is applied to decolorize many different dyes including

basic dyes (Methylene blue), acid dyes (Methyl orange, Aniline blue), reactive dyes
(Reactive black), azoic dyes (Evans blue). The results show that in most cases, the
enzyme can decolorize about 50% of the dye after 24 hours of reaction.
To study the effect of intermediates on the efficiency of deionization, some

intermediates such as Syringaldehyde, HOBT, Vanaline are used.
use.
Previous studies also showed complex results for decolorization depending on
the type of intermediate and dye. rFolac1 showed poor dye decolorization in the
absence of an intermediate in the case of Bromothymol blue, Reactive black, Orange
II, Methylene blue, Crystal violet, Evans blue. This result is similar to some previous
studies such as Lac1, rLac2 and rLcc9 which also showed the decolorization
efficiency of the dyes Blue M-2GE brilliant red KD-8B, Reactive
brilliant red KM-8B, Reactive brilliant orange K-7R, acid red 6B, Alizarin red, Congo
red, Neutral red, Crystal violet low in the absence of intermediates [30], [128]. Outside
106
In addition, some cases show only, in the absence of mediators, the decolorization
effect of about 2-5% of dyes. RLcc9 decolorization of indigo carmine and 5 azo dyes
was significantly increased when methyl syringate was used as an intermediate, with
decolorization rates ranging from 71.9 ± 3.2% to 99.1 ± 1.6% for different dyes [127].
Besides, most molds such as Trametes sanguineus decolorize 18% of Congo red
color without intermediates [8]. Lcc2 and Lcc6 were unable to decolorize Methyl
orange, Crystal violet and Malachite green dyes without an ABTS intermediate [119].
The recombinant fungal laccase has also been widely applied for the purpose
of biological remediation of the medium. For example, the recombinant protein Lcc1
and Lcc3 from T. trogii, produced in P. pastoris , has proven to be a useful biopolymer
for the degradation of some pollutant dyes such as Indigo carmine, which The most
important are used to produce blue jeans [15], [19]. In addition, Darvishi et al. (2018)
also expressed and produced a recombinant laccase (Lcc IIIb) from T. versolor in Y.
lipolytica, demonstrating the dye's decolorization capacity.
phenol azo with more than 40% efficiency after 4 h of treatment [20]. Similarly, Wang
et al. (2018), expressing a laccase from the fungus Laccaria bicolor in P. pastoris and
A.thaliana, demonstrated the ability to decolorize more than 80% of Crystal violet dye
at laboratory scale, providing a new method for the decolorization of industrial
wastewater [12]. In another study, Balcázar-López et al. (2016) expressed the gene
encoding the laccase from P. sanguineus in T. atroviride; The recombinant laccase
maintains the same properties as the native enzyme, however the recombinant enzyme shows
ability to remove more than 90% of phenanthrene and benzo ÿ - pyrene present in
wastewater from biofuel industrial plants by laboratory studies [9]. A recent study by
Huy et al (2021) also expressed the laccase gene (FoLacc5) in P. pastoris, rFoLacc5
removed many dyes in such as
Brommothymol blue, Methylene blue, Methyl orange, Remazol brilliant blue R, Aniline
blue, Evans blue, Indigo carmine and Orange II. Especially, up to 93.99% and 91.63%
of two dyes, Malachite green and Aniline blue, were removed [40].
107
The difference in the efficiency of the decolorization process may be due to the
difference in the molecular structure of the dye [40]. Laccase exhibits very high efficiency
in decolorization of the anthraquinone dye RBBR, similar to the purified laccase from P.
sanguineus [74]. In contrast, the recombinant laccase purified from P. methanolica
required 16 h to degrade about 90% of RBBR under laccase activity higher than 25 U/mL [32].
In the presence of intermediates, the decolorization of Indigo carmine by laccase can be
greatly enhanced [22]. However, many azo dyes are not laccase substrates, and their
decolorization depends on the presence of several intermediates. For substances such
as phenol aldehydes, ketones, acids and esters involving three lignin units, one of the
best intermediates is acid p.
coumaric, vanillin, acetovanillone, methyl vanillate, especially syringaldehyde and
acetosyringone. The last two compounds are potential mediators for industrial
applications as they support the highest decolorization rates in only 5 to 30 minutes
depending on the type of dye being treated [14].
From the above results, it can be seen that the need for intermediates, the
selection of intermediates depends not only on the characteristics of the dyes but also
on the properties of the enzymes that we used to treat the dyes. physical.
108
CONCLUSIONS AND RECOMMENDATIONS
1. CONCLUSIONS
From the research results, we draw the following conclusions: 1. A strain of
F. oxysporum with strong laccase biosynthesis ability has been isolated.
reached 186 U/mL after 12 days of fermentation on the medium supplemented with straw as a carbon source.
2. Successfully cloned Folac1, Folac2, Folac3 and Folac4 genes into vector
pGEM ® T-Easy. The genes have been successfully nucleotide sequenced and registered on the
Genbank gene bank with code: Folac1 (MT362052); Folac2 (KY825237);
Folac3 (KY825238) and Folac4 (KY825239).
3. Successful transfer of gene encoding laccase 1 (Folac1) in recombinant P. pastoris . The
presence of Folac1 in the genome of P. pastoris was confirmed by PCR. rFolac1 was purified from
the culture medium of recombinant P. pastoris strain with a molecular weight of about 76 kDa.
4. The recombinant P. pastoris X33 strain had the highest laccase biosynthesis when cultured
at 25oC, shaking speed 210 rpm, initial cell density OD600 = 1. Specific enzyme activity was 66538
U/mg after 4 days of culture and the protein concentration was 0.9 µg/µL. The recombinant laccase
has an optimal temperature of 55oC, an optimal pH of 4.5, Cu2+ ions increase enzyme activity while
Fe2+ ions inhibit laccase activity.
5. Recombinant laccase has the ability to decolorize about 50% of dyes after 24 hours of
reaction. In particular, Folac1 can decolorize 82.7% and 88.4% in the case of
Indigo carmine and Remazol brilliant blue R. Compared with the decolorization activity in F.
oxysporum HUIB02, recombinant Folac1 showed a significant improvement of 38.87%, 13.93%,
40.44% and 17.54% in the case of Crystal violet, Aniline blue, Evans blue and
Orange II.
2.
RECOMMENDATIONS 1. Optimization and production of rFolac1 at fermenter scale 5 L and larger in order to
increased recombinant enzyme production.
2. Test the application of recombinant laccase under laboratory and practical conditions to
treat many contaminant compounds such as heavy metals, antibiotics (in medical wastewater),
synthetic dyes .
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PUBLICATIONS RELATED TO THE THESIS
1. Dang Thi Thanh Ha, Le Kim Tuan, Dinh Thi Kim Thien, Pham Thi Ngoc Lan, Hoang Tan
Quang, Tran Thuy Lan, Nguyen Duc Huy (2018), Line creation and solution
gene sequence of laccase 3 (Folac3) from Fusarium oxysporum, Journal of Science, Hue
University. Episode 127, No. 1C, 2018; ISSN
1859-1388. 2. Dang Thi Thanh Ha, Duong Thi Hai Ninh, Thi Le Kim Tuan, Do Tran Huong
Duyen, Le My Tieu Ngoc, Pham Thi Ngoc Lan, Nguyen Duc Huy (2020), Isolation of the gene
encoding laccase4 from Fusarium oxysporum , Journal of Science, Tay Nguyen University. Issue
40, February 2020, ISSN: 1859-4611.
3. Nguyen Duc Huy, Dang Thi Thanh Ha, Kuan Shiong Khoo, Pham Thi Ngoc
Lan, Hoang Tan Quang, Nguyen Hoang Loc, Seung-Moon Park, Ashokkumar
Veeramuthu, Pau Loke Show (2020), Synthetic dyes removal by Fusarium
oxysporum HUIB02 and stimulation effect on laccase accumulation, Enviroment
Technology & Innovation 19: 101027.
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APPENDIX
Appendix 1: Sample pictures
Figure 1.1. Specimens were collected in Thua Thien Hue province.
Appendix 2: Research Diagram
Isolation of fungal strains with laccase . activity
Investigation of laccase . activity

126
Determination of laccase enzyme activity

Appendix 3. Culture media

Table 1. Isolation media composition
127
Wall Environment
part PDA PD
Potato 200 g
Dextrose 20 g
Distilled water 1L
Agar 15 g
Table 2. Components of screening medium
Wall Environment
part MF1 MF2 MF3 MF4
KH2PO4: 0.2 g; MgSO2.7H2O: 0.05 g, CaCl2.2H2O: 0.01 g
CuSO4.5H2O: 80 mg; Na2MoO4.2H2O: 50 mg, MnSO4.H2O: 33

BSM
mg
ZnSO4.7H2O: 43 mg, FeSO4.7H2O: 50 mg
Straw powder ten% 5%
Wood flour ten% 5%
Distilled water 1L
Table 3. LB . environment composition
Ingredient Environment
Liquid LB Snake LB
NaCl 10 g
Yeast extract 5g
128
Trypton 10 g
Distilled water 1L
Bacto agar 15 g
Table 4. YD . media composition

Liquid LB Snake LB
Yeast extract 5g
Pepton 10 g
Distilled water 1L
Bacto agar 15 g
Table 5. YDP . media composition
YPD liquid YPDS YDP solid YPM
Yeast extract 10 g
Pepton 20 g
Distilled water 1L
Dextrose 20 g
Bacto agar 0g 20 g
Sorbitol 1 US
Methanol 2%
Appendix 4. Bradford's BSA standard curve equation
129
2.5
y = 0.0194x + 1.442
R = 0.9976
2
1.5
value
OD
first
0.5
0
0 5 ten 15 20 25 30
BSA concentration (µg/mL)
Figure 1. Bradford BSA standard curve [14]

Appendix 5. DNA and protein markers used in the study
Figure 1. PreciGene 1 kb DNA Ladder Figure 2. GeneRuler 1 kb Plus DNA
Ladder
(BioPioneer)
(Thermo Scientific)
130
Figure 3. GeneRuler 1 kb DNA Ladder Figure 4. 100 bp PCR Molecular Ruler
(Thermo Scientific) (Bio-Rad)
Figure 5. PageRuler™ Prestained Protein Ladder, 10 to 180 kDa (Thermo

Scientific)
131
Appendix 6: Results of registration of nucleotide sequences of Fusarium oxysporum . strain
Fusarium oxysporum strain HUIB02 small subunit ribosomal RNA gene, partial sequence; internal
transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete
sequence; and large subunit ribosomal RNA gene, partial sequence
GenBank: KX388184.1
LOCUS KX388184 544 bp DNA linear PLN 27-NOV-2016
DEFINITION Fusarium oxysporum strain HUIB02 small subunit ribosomal RNA gene, partial sequence; internal
transcribed spacer 1, 5.8S ribosomal RNA gene, and
internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence.
ACCESSION KX388184
VERSION KX388184.1
KEYWORDS .
SOURCE Fusarium oxysporum
ORGANISM Fusarium oxysporum
Eukaryota; Fungi; Dikarya; Ascomycota; Pezizomycotina;
Sordariomycetes; Hypocreomycetidae; Hypocreales; Nectriaceae;
Fusarium; Fusarium oxysporum species complex.
REFERENCE 1 (bases 1 to 544)
AUTHORS Huy,ND, Be,NT, Tien,NTT and Huyen,LTD
TITLE High laccase production from Fusarium oxysporum HUIB02 on rice straw
medium
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 544)
AUTHORS Huy,ND, Be,NT, Tien,NTT and Huyen,LTD
TITLE Direct Submission
JOURNAL Submitted (14-JUN-2016) Department of Molecular Biology, Institute of Biotechnology - Hue University,
Ngoc Anh, Phu Thuong, Phu Vang, Hue, Thua Thien Hue 530000, Vietnam
COMMENT ##Assembly-Data-START##
Sequencing Technology :: Sanger dideoxy sequencing
##Assembly-Data-END##
FEATURES Location/Qualifiers
source 1..544 /
organism="Fusarium oxysporum" /
mol_type="genomic DNA" /
strain= "HUIB02" /
db_xref="taxon:5507"
misc_RNA <1..>544 /
note="contains small subunit ribosomal RNA, internal
transcribed spacer 1, 5.8S ribosomal RNA, internal
transcribed spacer 2, and large subunit ribosomal RNA"
ORIGIN
2 tcgatgaaga acgcagcaaa atgcgataag 241 taatgtgaat tgcagaattc
agtgaatcat cgaatctttg aacgcacatt gcgcccgcca 301 gtattctggc gggcatgcct
gttcgagcgt catttcaacgtgttcaacgtgc ctcaagt gag cttccatagc 421 gtagtagtaa
aaccctcgtt actggtaatc gtcgcggcca cgccgttaaa ccccaacttc 481 tgaatgttga
cctcggatca ggtaggaata cccgctgaac ttaagcatat caataagcgg 541 agga
//
132
Figure 1: Nucleotide sequence registration results of Fusarium oxysporum . strain
Appendix 7. Nucleotide sequences of genes encoding laccase
ATGACGAAGCTATCCCTGACACCTTTCTGTTCTCTCTCATTTACCATCTTACTCAAGCTCCA
GGGTTCTGAACCTTTGCCCAAGATGGGTTTCTACAGCGCGTGCAAGACGTTCATCGTCTAC
ACCGTTACGTTCTTCTCACTACCATTTCACGAGCATATCGGAGGTGACTCTGAGCAACCTGT
CTTGTCTTTCGAAAATGGCCAGATCTCACAAAACTATCCGAAGTTCCCAGCACCAAATGGC
CCCGATTCACCTGATGAAGACTTCACTTGCAAGTATCCCGAGCTTGGAAATGACTGGAAAC
CATGCTCTACCCCGGAAAACCGACACTGCTGGCTGAAGAGCTCTGATGGACACAATTTCAG
TATTCATACTGACTATGAGACTATGTATCCACCTGGGGTGCTTCGAGAGTATTGGCTTGTGG
TTGACAACAAGACTATCAACGGCGACGGCATCGACAACCCGTATGGAAAGGTCTTCAACC
AAACCTACCCCGGTCTATGGATCAAAGCCTGCTGGGGTGATCTCATCAGAGTTCACGTCAC
AAATAAACTGCGATATAACGGCACTACAATCCATTGGCATGGTACCAGGCAGAACGGAAC
CATGGAGATGGATGGAGTCAACGGTGTCACTCAATGTCCCATTGCCCCAAATGATACCTTC
ACATATGAGCTTCGTGCCCTTCAGTATGGTTTCTTCTTGGTATCATCATAGTCACTACTCTCTACA
ATACGCCGACGGTCTTGCAGGACCTATAACCATTTTTGGACCGTCATCAGCTCACTATGAT
GAAGCGAAGGATCCTATTCTTATCACCGATTGGAACCCATCGAAGTGCCTTTCAGGAGTGGG
AGAGAGAACTGACCGGTCTCCCTACCAGGCCCCAGATGAATAGCATATTGATGAATGGAA
TTGGAAACTTTGCCGGCAGCTATCCGCGCGAACGTTACAACATGACCGTAACCAAGGGCA
GGAAATATCTCCTTCGTATCATCAACACATCTGTCGATACCACATTCCTTTTCAGTATCGAC
AATCACCACTTTGAGGTCATGAGCTCCGACTTTGTACCCATTCACCCCTATACAGTAGACC
ATATCCTCGTCGGAATCGGCCAAAGATATCATGTTGTTCTTCATGCCAACCCTAGAAACGA
CACCAAATTTCCCGCGTCAGATAATGGAAACTATTGGATCCGCACAGTTCCCGGAGATGGT
TGCAAGGGTTTTGAAGATGGTAACGAGCCTGATGAACGGCAAGGTATTCTTAGATATGAAC
CTGTGAGCACAGAGGTACCTCAGACCTGGAGAGGCCCGTGGAACAAGACTTGCAGTGATG
AGAAGTATGAGAACTTGAAGCCTGTCCTGCCATGGTCGATCCCCCCTGTTGAACTCTATAG
TAGGACAAAAGCAAGGATTCTAAAACTTGGCCTGCAGCAGGAAAAGAATAGGCCTCATCT
GGGAGATAAGTTCAGCTGGTGGGCTTTCGGCGCACAGCCTCTCTGGCTCAACTTCTCGGAG
CCTACTATCACTTTACTTGACGAAAAAGAAAGAATGGCCTGGCGATTATGTCATTGTTCCAG
CAGAGAACAGAGGCGGATGGGTCTACCTTGTCATCACGGCTCCAGCAACTGAGGAGCTTG
GGAATAACAGGACATTCATCTCGCTTGCTCACCCACTTCATCTTCATGGTCATGACTTTGCT
CTTCTCGCTCAAGGTTCAGACTCTTCGCAGATTGATGACCCAGATAACCCCATCACACTCA
AATTCGACAACCCGCCTCGTCGAGACGTAGCACTGATCCCGGCTGGAGGCTACCTCATAGT
CGCCTTCAAGGCTGATAATCCTGGCTCTTGGTTGTTCCACTGCCATATTGCCTGGCATGCTT
CAAGTGGGTTGGCATTGCAGATCATGGAGCGTGAAGAAGATCTGAGGAAGATGATGACTC
CGGAGAAATTAAAGCAGGTCAATGAAGGCTGTAAGAAGTGGAAAGATTGGTATGGCGACC
GGAAGAATCACTGGAACCCTGTCGGCATATTCCAGGACGATTCTGGTGTTTGA
Figure 1. Nucleotide sequence of the Folac1 . gene
133
HUIB02 MTKLSLTPFCSLSFTILLKLQGSEPLPKMGFYSACKTFIVYTVTFFSLPFHEHIGGDSEQ 60
XM_018392545.1 MTKLSLTPFCSLSFTILLKLQSSEPLPKMGFYSACKTFVVYTVTFFSLPFHEHIGGDSEQ 60
XM_031207509.1 MTKLSLTPFCSLSFTILLKLQSSEPLPKMGFYSACKTFIVYTVTFFSLPFHEHIGGDSEQ 60
XM_031188070.1 ----------------------------MGFYSACKTFIVYTVTFLSLPFHEHIGGDSEQ 32
XM_031231991.1 ----------------------------MGFYNVCKSLIVYTATFFSLPLHEHIGDDSEQ 32
XM_023580876.1 ----------------------------MGFYNVCKSLIAYTATFFSLPLHEHTGGDSEQ 32
****..**:::.**.**:***:*** *.****
HUIB02 PVLSFENGQISQNYPKFPAPNGPDSPDEDFTCKYPELGNDWKPCSTPENRHCWLKSSDGH 120

XM_018392545.1 PVLSFENGQISQNYPKFPAPNGPDSPDEDFTCKYPELGNDWKPCSTPENRHCWLKSSDGH 120
XM_031231991.1 PVLSFENDRISQNYPKFPAPNGPDSPDEDFICKYPELGNDWKSCSTPENRHCWLKSSDGH 92
XM_023580876.1 PVLSFENDQISQNYPKFPAPNGPDSPDEDFICKYPELGNDWKSCSTPENRHCWLKSSDGH 92 *******.:********************* ******** *** *****************
HUIB02 NFSIHTDYETMYPPGVLREYWLVVDNKTINGDGIDNPYGKVFNQTYPGLWIKACWGDLIR 180

XM_018392545.1 NFSIHTDYETMYPPGVLREYWLVVDNKTINGDGIDSPYGKVFNQTYPGPWIKACWGDLIR 180
XM_031207509.1 NFSIHTDYETMYPPGVLREYWLVVDNKTINGDGIDNPYGKVFNQTYPGPWIKACWGDLIR 180
XM_023580876.1 NFSIHTDYETMYPPGVLREYWLVVDNKTINGDGIDNPYGKVFNQTYPGPWIKACWGDLIR 152 ***********************************.** ********** ***********
HUIB02 VHVTNKLRYNGTTIHWHGTRQNGTMEMDGVNGVTQCPIAPNDTFTYELRALQYGSSWYHS 240

XM_018392545.1 VHVTNKLRYNGTTIHWHGIRQNGTMEMDGVNGVTQCPIAPNDTFTYEFRALQYGSSWYHS 240
XM_031231991.1 VHVTNKLRYNGTTVHWHGIRQNGTMEMDGVNGVTQCPIAPEDTFTYEFRALQYGSSWYHS 212
XM_023580876.1 VHVTNKLRYNGTTVHWHGIRQNGTMEMDGVNGVTQCPIAPEDTFTYEFRALQYGSSWYHS 212 *************:**** *********************:********:*
***********
HUIB02 HYSLQYADGLAGPITIFGPSSAHYDEAKDPILITDWNHRSAFQEWERELTGLPTRPQMNS 300

XM_018392545.1 HYSLQYADGLAGPITIFGPSSAHYDEAKDPILITDWNHRSAFQEWERELTGVPTRPQMNS 300
XM_031231991.1 HYSLQYADGLAGPITIFGPSSAHYDEAKDPILITDWNHRSAFQEWERELTGVPTRPEMNS 272
XM_023580876.1 HYSLQYADGLAGPITIFGPSSAHYDEAKDPILITDWNHRSAFQEWERELTGVPTRPEMNS 272
************************************************** *:****:***
HUIB02 ILMNGIGNFAGSYPRERYNMTVTKGRKYLLRIINTSVDTTFLFSIDNHHFEVMSSDFVPI 360

XM_018392545.1 ILMNGIGNFAGSFPRERYNMTVTKKKYLLRIINTSVDTTFLFSIDNHHFEVMSSDFVPI 360
XM_031231991.1 ILMNGIGNFAGSFPRERYNITVTKKKKYLLRIINTSVDTTFVFGIDNHYFEVMSSDFVPI 332
XM_023580876.1 ILMNGIGNFAGSFPRERYNITVTKKKKYLLRIINTSVDTTFLFGIDNHYFEVMSSDFVPI 332 ************:******:*****:****************:*.** **:***********
HPYTVDHILVGIGQRYHVVLHANPRNDTKFPASDNGNYWIRTVPADGCKGFEDGNEPDER 420
XM_018392545.1 HPYTVDHILVGIGQRYHVVLHANPRNDTKFPASDNGNYWIRTVPADGCKGFEDGNEPDER 420 HPYTVDHILVGIGQRYHVVLHANPRNDTKVPADVLKGFNDGC
XM_031207509.1 YTVDHILVGIGQRYHVVLHAKPRNDTKFPASNNGNYWIRTVAADGCKGFEDGNEPDER 392 **********************:*** *******:****************************
XM_031188070.1
XM_031231991.1
XM_023580876.1
HUIB02 QGILRYEPVSTEVPQTWRGPWNKTCSDEKYENLKPVLPWSIPPVELYSRDKSKDLKLGLQ 480

XM_018392545.1 QGILRYEPVSSEVPQTWRGPWNKTCSDEKYENLKPVLPWSIPPVELYSRDKSKDLKLGLQ 480
XM_031207509.1 QGILRYEPVSTEVPQTWRGPWNKTCSDEKYENLKPVLPWSIPPVELYSRDKSKDLKLGLQ 480
XM_031188070.1 QGILRYEPVSTEVPQTWRGPWNKTCSDEKYENLKPVLPWSIPPVELYSRDKSKDLKLGLQ 452
XM_031231991.1 QGILRYQPVSTEVPQTWRDNWTKDCTDEKYENLKPILPWSIPTVKLDERDRSKDLELGIE 452
XM_023580876.1 QGILRYQPVSTEVPQTWRDNWTKDCTDEKYENLKPILPWSIPTVKLDERDRSKDLELGIE 452 ******:***:*******. *.* *:*********:****** *:* .**:****:**::
HUIB02 QEKNRPHLGDKFSWWAFGAQPLWLNFSEPTITLLDEKKEWPGDYVIVPAENRGGWVYLVI 540

XM_018392545.1 QEKNRPHLGDKFSWWAFGAQPLWLNFSEPTITLLDEKKEWPGDYVIVPAENRGGWVYLVI 540
XM_031231991.1 KVKDRPQAGDRFSWWAFGERPLWLDFSNPTITLLEERKEWPDDYVIVPAENRDGWVYLVI 512
XM_023580876.1 KVKDRPHAGDRFSWWAFGERPLWLDFSNPTITLLEERKEWPDDYVIVPAENRDGWVYLVI 512
: *:**: **:******* :****:**:******:*:****.**********. *******
HUIB02 TAPATEELGNNRTFISLAHPLHLHGHDFALLAQGSDSSQIDDPDNPITLKFDNPPRRDVA 600

XM_018392545.1 TAPATEELGNNRTFISLAHPLHLHGHDFALLAQGSDSSQIDDPDNPITLKFDNPPRRDVA 600
XM_031207509.1 TAPATKELGNNRTFISLAHPLHLHGHDFALLAQGSDSSQIDDPDNPITLKFDNPPRRDVA 600
XM_031188070.1 TAPATEELGNNRTFISLAHPLHLHGHDFALLAQGSDSSQIDDPDNPITLKFDNPPRRDVA 572
XM_031231991.1 TAPSLDRIGTNKTFRSLAHPLHLHGHDFALLAQGTNSSQINDPNNPVTLKFDNPPRRDVA 572
XM_023580876.1 TAPSLDRIGTNKTFRSLAHPLHLHGHDFALLAQGTNSSQINDPNNPVTLKFDNPPRRDVA 572
***: ..:*.*:** *******************::****:**:**:***** ********
HUIB02 LIPAGGYLIVAFKADNPGSWLFHCHIAWHASSGLALQIMEREEDLRKMMTPEKLKQVNEG 660

XM_018392545.1 LIPAGGYLIVAFKADNPGSWLFHCHIAWHASSGLALQIMEREEDLRKMMTPEKLKQVNDG 660
XM_031207509.1 LIPAGGYLIVAFKADNPGSWLFHCHIAWHASSGLALQIMEREEDLRKMMTPKKLKQVNDG 660
XM_031188070.1 LIPAGGYLIVAFKADNPGSWLFHCHIAWHASSGLALQIMEREEDLRKMMTPEKLKQVNDG 632
XM_031231991.1 LIPAGGYLIVAFKADNPGSWLFHCHIAWHASSGLALQIMESEEDLRKMTSEKLKQVNDG 632
XM_023580876.1 LIPAGGYLIVAFKADNPGSWLFHCHIAWHASSGLALQIMEREEDLRKMMTSEKLKQVNDG 632
**************************************** ********* : ******:*
Figure 2. Comparative amino acid sequence inferred Folac1 gene of F. oxysporum HUIB02
strain and 5 other fungal strains. Differences in aa between strains are represented by a
black background.
134
ATGGCTCTCATAGAGCGAGTATGGCACGCCTGCGTCAGTATAGTCGCATGGCTGACAATGTG
GCCCACCT
CCTACCAACATCCCCTCCGCCCCAATCATCCTCACACTCACATTGAAGATCCCAGCCCTGACT
TCCCCAT
CTTCAATCCCCCGCCAGGTGATCATGAATTTCTCTGCGAGTACCCAGAAATGACAGGTTTCGT
GCAATGT
TCAATTCCTGAGAATCGAGAATGTTGGCTTAGACATCCGGATGGGCGAGAGTTCAACATCCA
TACAAACT
ATGAGAACTTTGCACCGAAGGGTATTATGAGGCATTATACACTTAATGTTACTGAGAGTTGGT
ATAATGC
TGATGGGCAGAACTTTACCGAGGCGAAGTTGTTCAATGGGGAGTATCCTGGGCCTTGGCTTG
AGGCTTGT
TGGGGTGATGTGAGTACTCTGGCCCGTGGGGTTTGAGAAAGGGACTAAGTGTTGTGCAGACC
TTCAATAT
CACGGTGATCAATAGTATGAAGCGGAACGGCACGAGTATTCACTGGCATGGTATTCGACAGA
ACCAAACG
ATGGACATGGATGGTGTCAACGGCATAACTCAATGTCCAATTGCGCCAGGGGATAGCTTCAG
CTACATCT
TCAATACGACGCAGTACGGGACGTCTTGGTATCATAGCCACTACTCTGTGCAGTATGCAGAT
GGTCTTCA
GGGCCCAATCGTAAGCCAAACCGCGGTGCTCAGCCAAAATCGCGCTAACGATGCAGACAATT
CATGGTCC
CCAGTCAGCTCCCTACGATGCAACCAAGAGGCCATTACTTATGACAGATTGGTGTTAGTTGCC
CCCAAGT
AGGCTGCATGGCCCATACTGACTGAACATACAGCTCACGAGAGTGCATTCAGACTACTCTTC
CCTGGCTC
CCAATTCTCCAACAAGACCATCCTTCTCCAACGGCGCAGGCAACGTCTCCCACTACGGCTACA
CCCCAACT
CTCCCTGTCCCTGATAACTATGAATTATACTTCAATAAAACTCCAACAGATAAGCCAAGTCGC
CCAAAGA
GATATTTGCTCCGCCTGATCAATACTTCATTTGACTCAACACTCGTCTTCTCCATTGATAACCA
CTGGCT
TCAAATCGTCACTTCGGACTTTGTCCCTATTGAGCCGTACTTCAACACCTCAGTCTTGATAGG
AATTGGT
CAAAGGTACAACGTCATTGTCGAAGCGAATCCTCTTGCCGGTGATGTCAACGAGATCCCAGA
AGATGGAA
ACTTCTGGATCAGGACTTGGGTCGCTGATGCATGTGGTATAGCCCCAGGAGGAGACGGTTAC
GAAAAGAC
CGAATCCTACGCTACAACTACACTGACAAGGCTCTTCCCTCATCTCAGCCTTGGGTCAACAT
CAGCAAA
GCGTGCTCAGATGAGACATATACTTCGCTTCGACCGAAGATACCATGGTACATTGGTCCAGCT
GCCAATG
CTCAAGCGGCGGATCAGACCGGTGAGCGGTTCAATGTTACTTTTGATCCGAATGCCAAGAAT
ACACCTGA
ATTCCAGGAGGAGTATCCGGTTGCTACGTTTGGTCTCCAGAGACCTGGTCAGAACTTTAGACC
ACTGCAG
ATTAATTACTCTGATCCCGTTATGTTTCACTTGGATGAGCCGAGGGATACTTACCCACCGAAG
TGGGTGG
TGATTCCTGAGGATTATACTGAGAAAGAATGGGTATGCGTCAGACGCTTTCAAATGTCTTCGG
GTATCAT
GACTAACCCATCATAGGTCTACTTTGTTCTTACGATAGAAGGCATTAGTGCCCGCACCGGCGCT
CATCCAG
135
TAAGCATCCAACCCCTGCCACATGTTCCCTAGCTAACCCTATTCAGATCCACCTTCACGGGCA
TGACTTT
GCCCTTCTGCAACAAGAAGAGAACCAAACCTATGACCCCAGCAGACTCAACTTGAAGCTTGA
CACCCAC
CAAGGCGTGATGTCGTCCTACTTCCCCGCAATGGCTTCGTAGTTATTGCTTTCAAGGCAGATA
ACCCAGG
TATCTGGCTGATGCACTGCCACATTGCGCGCCATGCCTCCGAAGGTTTGGCTATGCAAGTCCT
GGAGAGA
CAGGGAGATGCCAATGAGTTATTCCCTGTTGGTTCGACGAATATCATTGAGGCGGAAAGGGT
TTGTAAGA
ACTGGAAGACGTGGATGGATGGTGAGAAGGATTTCTTTGAAGGTGATTCTGGTATTTAA
Figure 3. Folac2 nucleotide sequence . The intron noncoding region sequences are
annotated with white lettering on a black background.
136
XM_031228032.1 XM_018901706.1 MALIERVWHACVSIVAWLTMWPTSPSTSYQHPLRPNHPHTHIEDPSPGFPIFHPPPGDHE 60

XM_031192261.1 XM_031210768.1 MALIERVWHACVSIVAWLTMWPTSPSTSYQHPLRPNHPHTHIEDPSPGFPIFHPPPGDHE 60
HUIB02 XM_018393137.1 MALIERVWHACVSIVAWLTMWPTSPSTSYQHPLRPNHPHTHIEDPSPGFPIFNPPPGDHE 60
MALIERVWHACVSIVAWLTMWPTSPSTSYQHPLRPNHPHTHIEDPSPGFPIFHPPPGDHE 60
MALIERVWHACVSIVAWLTMWPT----SYQHPLRPNHPHTHIEDPSPDFPIFNPPPGDHE 56
MALIERVWHACVSIVAWLTMWPTSPSTSYQHPFRPNHPHTHIEDPSPGFPIFNPPPGDHE 60
*********************** *****:**************.****:*******
XM_031228032.1 XM_018901706.1 FLCEYPEMTGFVQCSIPENRECWLRHPDGREFNIHTNYENFAPKGIMRHYTLNITESWYN 120

XM_031192261.1 XM_0312107681 FLCEYPEMTGFVQCSIPENRECWLRHPDGREFNIHTNYENFAPKGIMRHYTLNITESWYN 120
FLCEYPEMTGFVQCSIPENRECWLRHPDGREFNIHTNYENFAPKGIMRHYTLNVTESWYN 120
FLCEYPEMTGFVQCSIPENRECWLRHPDGREFNIHTNYENFAPKGIMRHYTLNITESWYN 120
HUIB02 FLCEYPEMTGFVQCSIPENRECWLRHPDGREFNIHTNYENFAPKGIMRHYTLNVTESWYN 116
XM_018393137.1 FLCEYPEMTGFVQCSIPENRECWLRHPDGREFNIHTNYENFAPKGIMRHYTLNVTESWYN 120 ******************************************** *****:******
XM_031228032.1 XM_018901706.1 ADGQNFTEAKLFNGEYPGPWLEACWGDTFNITVINSMKRNGTSIHWHGIRQNQTMDMDGV 180

XM_031192261.1 XM_031210768.1 ADGQNFTEAKLFNGEYPGPWLEACWGDTFNITVINSMKRNGTSIHWHGIRQNQTMDMDGV 180
HUIB02 XM_018393137.1 ADGQNFTEAKLFNGEYPGPWLEACWGDTFNITVINSMKRNGTSIHWHGIRQNQTMDMDGV 180
ADGQNFTEAKLFNGEYPGPWLEACWGDTFNITVINSMKRNGTSIHWHGIRQNQTMDMDGV 180
ADGQNFTEAKLFNGEYPGPWLEACWGDTFNITVINSMKRNGTSIHWHGIRQNQTMDMDGV 176
ADGQNFTEAKLFNEEYPGPWLEACWGDTFNITVINSMKRNGTSIHWHGIRQNQTMDMDGV 180
************* ********************************* *********
XM_031228032.1 XM_018901706.1 NGITQCPIAPGDSFSYIFNTTQYGTSWYHSHYSVQYADGLQGPITIHGPQSAPYDATKRP 240

XM_031192261.1 XM_0312107681 NGITQCPIAPGDSFSYIFNTTQYGTSWYHSHYSVQYADGLQGPITIHGPQSAPYDATKRP 240
NGITQCPIAPGDSFSYIFNTTQYGTSWYHSHYSVQYADGLQGPITIHGPQSAPYDATKRP 240
NGITQCPIAPGDSFSYIFNTTQYGTSWYHSHYSVQYADGLQGPITIHGPQSAPYDATKRP 240
HUIB02 NGITQCPIAPGDSFSYIFNTTQYGTSWYHSHYSVQYADGLQGPITIHGPQSAPYDATKRP 236
XM_018393137.1 NGITQCPIAPGDSFSYIFNTTQYGTSWYHSHYSVQYADGLQGPITIHGPQSAPYDATKRP 240
************************************************** **********
XM_031228032.1 XM_018901706.1 LIMTDWSHESAFRLLFPGSQFSNKTILLNGAGNVSHYGYTPTLPPIPDNYELYFNKTPTDK 300

XM_031192261.1 XM_031210768.1 LLMTDWSHESAFRLLFPGSQFSNKTILLNGAGNVSHYGYTPTLPPIPDNYELYFNKTPTDK 300
HUIB02 XM_018393137.1 LLMTDWSHESAFRLLFPGSQFSNKTILLNGAGNVSHYGYTPTLPVPDNYELYFNKTPTDK 300
LLMTDWSHESAFRLLFPGSQFSNKTILLNGAGNVSHYGYTPTLPVPDNYELYFNKTPTDK 300
LLMTDWSHESAFRLLFPGSQFSNKTILLNGAGNVSHYGYPTLPPVPDNYELYFNKTPTDK 296
LLMTDWSHESAFRLLFPGSQFSNKTILLNGAGNVSHYGYPTLPPVPDNYELYFNKTPTDK 300 *:****************************************:*** ************
XM_031228032.1 XM_018901706.1 PSRPKRYLLRLINTSFDSTLVFSIDNHWLQIVTSDFVPIEPYFNTSVLIGIGQRYNVIVE 360

XM_031192261.1 XM_031210768.1 PTRPKRYLLRLINTSFDSTLVFSIDNHWLQIVTSDFVPIEPYFNTSVLIGIGQRYNVIVE 360
HUIB02 PSRPKRYLLRLINTSFDSTLVFSIDNHWLQIVTSDFVPIEPYFNTSVLIGIGQRYNVIVE 360
PSRPKRYLLRLINTSFDSTLVFSIDNHWLQIVTSDFVPIEPYFNTSVLIGIGQRYNVIVE 360
PSRPKRYLLRLINTSFFDSTLVFSIDNHWLQIVTSDFVPIEPYFNTSVLIGIGQRYNVIVE 356
XM_018393137.1 PSRPKRYLLRLINTSFDSTLVFSIDNHWLQIVTSDFVPIEPYFNTSVLIGIGQRYNVIVE 360
*:******************************************** **********
XM_031228032.1 XM_018901706.1 Anplagdvnqipddnfwrirtwvadggiapgdgyktgilylryhalpssqpwvni 420 anplggdvneipddnfwwvadacgiapggegyktgilryhalpssqpwdni 420

XM_031192261.1 XM_031210768.1 anplagdvneipedgnfwwrvadgdgdagykthtlrythndkrdkthidenpdhidenpthidenpthiddhiddhiddhidth Rynhtdkalpssqpwvni 420 anplagdvneipednfwwvadacgiapggdgytgyktgilrynytlrytkalpssqpwvni
HUIB02 XM_018393137.1 416 anplagdvneipedgnfrirtwvadacgiapggdgykylrynhthathathtdkalpssqpwvni 420 *** *** *** *** *** *** *** *** *** ***:
************************************************************************************************************************************************************* ******::****************
XM_031228032.1 XM_018901706.1 SKACSDETYTSLRPKIPWYIGPAANAQAADGNGERFNVTFDPNAKNTPEFQEEYPVATFG 480

XM_031192261.1 XM_0312107681 SKACSDETYTSLRPKIPWYIGPAANAQ----NGERFNVTFDPNAKNTPEFQEEYPVATFG 476
SKACSDETYTSLRPKIPWYIGPAANAQAADQTGERFNVTFDPNAKNTPEFQEEYPVATFG 480
SKACSDETYTSLRPKIPWYIGPAANAQAADQTGERFNVTFDPNAKNTPEFQEEYPVATFG 480
HUIB02 SKACSDETYTSLRPKIPWYIGPAANAQAADQTGERFNVTFDPNAKNTPEFQEEYPVATFG 476
XM_018393137.1 SKACSDETYTSLRPKIPWYIGPAANAQAADQTGERFNVTFDPNAKNTPEFQEEYPVATFG 480
*************************** .****************************
XM_031228032.1 XM_018901706.1 LQRPGQNFRPLQINYSDPVMFHLDEPRDTYPPKWVVIPEDYTEKEWVYFVLTIEGISART 540

XM_031192261.1 XM_0312107681 LQRPGQNFRPLQINYSDPVMFHLDEPRDTYPPKWVVIPEDYTEKEWVYFVLTIEGISART 536
LQRPGQNFRPLQINYSDPVMFHLDEPRDTYPPKWVVIPEDYTEKEWVYFVLTIEGISART 540
LQRPGQNFRPLQINYSDPVMFHLDEPRDTYPPKWVVIPEDYTEKEWVYFVLTIEGISART 540
HUIB02 LQRPGQNFRPLQINYSDPVMFHLDEPRDTYPPKWVVIPEDYTEKEWVYFVLTIEGISART 536
XM_018393137.1 LQRPGQNFRPLQINYSDPVMFHLDEPRDTYPPKWVVIPEDYTEKEWVYFVLTIEGISART 540
************************************************** **********
XM_031228032.1 XM_018901706.1 GAHPIHLHGHDFALLQQEENQTYDPSRLNLKLDNPPRRDVVLLPRNGFVVIAFKADNPGI 600

XM_031192261.1 XM_031210768.1 GAHPIHLHGHDFALLQQEENQTYDPSRLNLKLDNPPRRDVVLLPRNGFVVIAFKADNPGI 596
HUIB02 GAHPVSIQPLPHVP---------------------------------------------------------------- 554
GAHPIHLHGHDFALLQQEENQTYDPSRLNLKLDNPPRRDVVLLPRNGFVVIAFKADNPGI 600
GAHPIHLHGHDFALLQQEENQTYDPSRLNLKLDNPPRRDDVVLLPRNGFVVIAFKADNPG-595
XM_018393137.1 GAHPIHLHGHDFALLQQEENQTYDPSRLNLKLDNPPRDVVLLPRNGFVVIAFKADNPG-599
****: :: ..
XM_031228032.1 XM_018901706.1 WLMHCHIARHASEGLAMQVLERQGDANKLFPAGSPNMIEAERVCKVWEAWMDGEKDFFEG 660

XM_031192261.1 XM_031210768.1 WLMHCHIARHASEGLAMQVLERQGDSNKLFPVGSPNMIEAERVCKVWETWMDGEKDFFEG 656
HUIB02 XM_018393137.1 --------------------------------------------------- ---------- 554
WLMHCHIARHASEGLAMQVLERQGDANELFPVGSPNMIEAERVCKDWKTWMDGEKDFFEG 660
--------------LAMQVLERQGDANELFPVGSTNIIEAERVCKNWKTWMDGEKDFFEG 641
--------------LAMQVLERQGDANELFPVGSPNIIEAERVCKNWKTWMDGEKDFFEG 645
XM_031228032.1 XM_018901706.1 DSGI 664

XM_031192261.1 XM_031210768.1 DSGI 660
HUIB02 XM_018393137.1 ---- 554
DSGI 664
DSGI 645
DSGI 649
Figure 4. Comparison of the amino acid sequence inferred by Folac2 gene of F. oxysporum
HUIB02 strain with 5 other fungal strains. The differences in aa of the strains are represented
by a black background.
137
ATGGTCGCTCACGATGAGAAGCCGATGCCTTGCTTGAGGAGTTTGAACTGCGAGAGGAG
GTCAAGGTCCGAGATGATCGAACCCCGCCTTCAGTGTGTCGCACGTGCGGTTGGAGATGG
ATAACAGCTCTTATCGTGGTCGCACTACTATCGGCAATCGGGGTCGTAGTGGTATTGCATG
GTCCGTTAAGCAACCAGTTGAGCGATGATGCTACCAGTCATCTTGGGTACCGACTCCATCC
TCAGAGGTCATGCATCACGTCCCCCAACAACACAATCTTTCAACTGGACTATTACAGCTGG
GACCAGATCACCAGATGGAGTTGAGAAAAGAGTGTACCTTGTCAACAGTAAGGTCCTCGT
CAAGTATGGTGCATACACTCACTGACTTTCGCAGATGAGTTTCCAGGACCCCTCATCGAG
GCCCGGTCTGGTGACCGACTTGTAATTCATATTCACAATGGCGTTCAAGATGAAGGTGTAT
CTCTTCATTGGCACGGCCTGCGCATGAAAGACCAGAACAGTGTGGACGGTGCTGTTGGGT
TTACACAATGTCCTATCGCTCCCGGTAGAAGTTTCACGTACAACTTCACCATTGGCGCTGA
AGACATGGGACCTTCTGGTGGCATCCCCATTCCGATGTTCAACGTGCCGACGGCCTTATG
GGGAGGTCTGATCGTACACTCACCAGATGAGATTGATTTGCAACGTGAAGAATACTTACT
CATGGTCGGCGACTGGTTTCACCAGAATCAAACGGAAGTACTACGCTGGTATGCTGATGC
GAGTAGTCGAGGGAATGAACCCGTCCCAGATTCATTGTTGGTCAATGGTCAGGGACGCTT
CAACTGCTCCATGGCTGTTCCTGCGAGACCAGTTGCTTGCTCTCAGGTCCAGTTTAGTGAC
CTCAAGCCCCTCATGATGTCTAGGAGCCAGAAGAAGGCCAGACTTAGAGTTGTCAACACA
GGCTCCGTCGCAGGCCTTTCCCTAAGAGCTGGTGGAGCAATCATTCGACCAGTTCGCGTG
GATGGAGGCTTCGCTGTCAAAGCTGAAGCTACTGAAACAGTTGGTAATCTCTATCCTGGT
GAGAGAGTTGACCTCGAGGTTGAGTGGAAGGGGAACCACGCGGGAGACCATCGGCTGAC
CGTTTATCTTGATGACGAGTGCGTTACTATCTGTTTCACTATTGGTAGAGATGGGGAGGCTA
ACCAGCTTGGCAGAAACTTTGGCTATCCCAACCCAGCACTTAACCCAACCCAAAGCTTCC
CAATGTTCAGCTCAAGCACAAAGGAATCATCCAATGAACCTGTTCCTCAGCCTTTGGAAC
AAGATGAGATCCAAGTCCTTGACTCGCATAATTCCAAAGCCGCAACGAAGGTATCGGACC
TCCCAGCAAAGGCAGAACAGACTATCCTGCTTTACGCTAAGGTTGAGAAGCTGGCGCACA
TGGACTATGCACCAGTCGGATTCATCAATCATACATCCTGGACGCCACAAACTCCACCTTT
ACTGGCTCAAAACCGGACTTCATGGGACGAGAACCAACTCATCCCATTTATCGGGATATC
AGACAGCAAACCGAAGAGAGTCGACATCGTCATCAACAACCTGGATGATGGCGCGCATC
CGTTTCATCTTCATGGACATTCATTCTACGTTCTATCATCATACCGAAATCCAGGTCGTGG
GAGCTGGGGCAGTTATAATCCGTACACAGACGAGGCGCCACCGAACGGCTTGAATCTTGA
GTTTCCGGTGGCGAAGGGATACTGTGAGTGTACCACGGCGCGGTCATGTCGTGTTGGCGTT
GGTGGCTGATAACCCGGGAATATGGGCGTTGCATTGCCACACGCTTGTTCACATGGCGCG
GGGTATGGCGATGGGGTTACGTGTTGGCGATATCGAGGATCCAGAGCATGTAGGCTCGGT
AGATGTGCGGGCGGCGGAGTTATGTGAGTAA
Figure 5. Folac3 gene nucleotide sequence isolated from F. oxysporum strain HUIB02.
The intron noncoding region sequences are annotated with white lettering on a black
background.
138
XM_018901654.1 MVAHDEEADALLEEFELREEVKVRDDRVLPLVCCTCGWRWIAALIVVALLSAIGVVVALH 60
XM_031228099.1 MVAHDEEADALLEEFELREEVKVRDARTPPSVCCTCGWRWIAALIVVALLSAIGVVVALY 60
XM_031210721.1 MVAHDEEADALLEEFELREEVKVRDDRTPPSVCRTCGWRWITALIVVALLSAIGVVVVLH 60
XM_031192310.1 HUIB02 MVAHDEEADALLEEFELREEVKVRDDRTPPSVCRTCGWRWIAALIVVALLSAIGVVVVLH 60
MVAHDEEADALLEEFELREEVKVRDDRTPPSVCRTCGWRWITALIVVALLSAIGVVVVLH 60
XM_018393180.1 MVAHDEEADALLEEFELREEVKVRDDRTPPSVCRTCGWRCMTALIVVALLSAIGVVVVLH 60
************************* *. * ** ***** ::****************.*:
XM_018901654.1 GLSSSQPSNDTTGHLGYRLHPQNHTSRPPTTLSFNWTITTGTRSPDGVEKQVYLVNDEFP 120

XM_031228099.1 GPSSSPPGNDTTGHLGYRLHPEDHAKRPPTTQYFNWTIAAGTKSPDGVEKRVYLVNNEFP 120
XM_031210721.1 GPLSNQLSDDATGHLGYRLHPQSHASRPPTQLSGNSFNWTITAGYTRVNFPDGVEKSGQLPTQLPSNWTITAGQLPSNNEQFP
XM_031192310.1 DATSHLGYRLHPQSHASRPPTTQSFNWTITAGTRSPDGVEKRVYLVNNEFP 120
HUIB02 GPLSNQLSDDATSHLGYRLHPQSHASRPPTTQSFNWTITAGTRSPDGVEKRVYLVNNEFP 120 * *. .:*:*.********:.*:.***** *****::**:*******:*****:**
XM_018393180.1 *
XM_018901654.1 GPLIEARSGDRVVIHVHNGLQDESVSLHWHGLRMKDQNNMDGAVGFTQCPIAPGRTFTYN 180

XM_031228099.1 GPLIEARSGDRIVIHVRNGLQDEGLSLHWHGLRMRYQNSMDGAVGFTQCPITPGTSFTYN 180
XM_031210721.1 GPLIEARSGDRLVIHVHNGVQDEGVSLHWHGLRMKYQNSMDGAVGFTQCPIAPGRSFTYN 180
XM_031192310.1 HUIB02 GPLIEARSGDRLVIHVHNGVQDEGVSLHWHGLRMKDQNSVDGAVGFTQCPIAPGRSFTYN 180
GPLIEARSGDRLVIHIHNGVQDEGVSLHWHGLRMKDQNSVDGAVGFTQCPIAPGRSFTYN 180
XM_018393180.1 GPLIEARSGDRLVIHVHNGVQDEGVSLHWHGLRMKDQNSVDGAVGFTQCPIAPGRSFTYN 180 ***********:***::**:***.:*********:
**. :************:** :****
XM_018901654.1 FTIGAEEYGTFWWHSHSDVQRADGLWGGLVVHYPHEVDIPQEDYLVMIGDWFHQNQTEVL 240

XM_031228099.1 FTIGAEEQGTFWWHSHSDVQRADGLWGGLVVHSPDEVDVPPEDYLLMIGDWFHRNQTEVL 240
XM_031210721.1 FTIGAEEHGTFWWHSHSHSDVQRADGLWGGLIVHSPDEIDLQREEYLLMVSDWFHQNQTEVL 240
XM_031192310.1 HUIB02 FTIGGEEHGTFWWHSHSDVQRADGLWGGLIVHSPDEIDLQRGEYLLMVGDWFHQNQTEVL 240
FTIGAEEHGTFWWHHPHSDVQRADGLWGGLIVHSPDEIDLQREEYLLMVGDWFHQNQTEVL 240
XM_018393180.1 FTIGAEEHGTFWWHSHSDVQRADGLWGGLIVHSPDEIDLQREEYLLMVGDWFHQNQTEVL 240
****.** ****** **************:** *.*:*: :**:*:.****:** ****
XM_018901654.1 RWYADASSRGNEPVPDSLLVNGKGRFNCSMVVPARPVACSQVQFSDLKPLMTSRSQNKVR 300

XM_031228099.1 GWYADASSRGNEPVPDSLLVNGRGRFNCSMAVPARPVVCSQVHLSDLKPLIMSGSLKKVR 300
XM_031210721.1 RWYADASSRGNEPVPDSLLLNGQGRFNCSMAVPARPVACSQVQFSDLKPLVMSRGQKKAR 300
XM_031192310.1 HUIB02 RWYADASSRGNEPVPDSMLVNGQGRFNCSMAVPARPIACSQVQFSDLKPLMMSRGQKKAR 300
RWYADASSRGNEPVPDSLLVNGQGRFNCSMAVPARPVACSQVQFSDLKPLMMSRSQKKAR 300
XM_018393180.1 RWYADASSRGNEPVPDSLLVNGQGRFNCSMAVPARPVACSQVQFSDLKPLMMSRGQKKAR 300
**************:*:**:*******.*****:.****::******: * . :*.*
XM_018901654.1 LRVVNTGSVAGLSLGVGRAILRPVRMDGGFAVKSEAAETVGTLYPGERVDLEVEWKRNHP 360

XM_031228099.1 IRVVNTGSVAGLSLRVGGAIIRPVRVDGGFAVKTEATETVGVLYPGERVDLEVEWKENYA 360
XM_031210721.1 LRVNAGSVAGLSLRVGGAIIRPVCVDGGFAVKAEATETVGILYPGERVDLEVEWKGNHA 360
XM_031192310.1 HUIB02 LRVVNTGSVAGLSLRVGGAIIRPVRVDGGFAVKAEATETVGILYPGERVDLEVEWKGNHA 360
LRVVNTGSVAGLSLRAGGAIIRPVRVDGGFAVKAEATETVGNLYPGERVDLEVEWKGNHA 360
XM_018393180.1 LRVVNTGSVAGLSLRVGGAIIRPVRVDGGFAVKAEATETVGILYPGERVDLEVEWKGNHA 360
:****:******** .* **:*** :********:**:**** ************ ** *:
XM_018901654.1 GDDWLTIYMDDENFGYPNLALNPTQSFPMFSSSTKGSFNEPVPQPLDEGAIQVLDSQNLK 420

XM_031228099.1 GDRWLTVYMDDENFGYPNPALHPTQSFPMFSSSAKGSFDEAVPQPLKQDEIQVLDSQNLK 420
XM_031210721.1 GDHRLTVYLDDENFGYPNPALNPTQSFPMFSSSTKGSSNEPVPQPLEQDEIQVLDSQNLK 420
XM_031192310.1 HUIB02 GDHRLTVYLDDENFGYPNPALNPTQSFPMFSSSTKEPSNEPVPQPLEQDEIQVLDSHNLK 420
GDHRLTVYLDDENFGYPNPALNPTQSFPMFSSSTKESSNEPVPQPLEQDEIQVLDSHNLK 420
XM_018393180.1 GDHRLTVYLDDENFGYSNPALNPTQSFPMFSSSTKESSNEPVPQALEQDEIQVLDSHNLK 420 ** **:*:******* * **:***********:* :* *** *. :.
******:***
XM_018901654.1 AAAKVSDLPPKAEQTILLYAKVEKLAHMDYAPVGFINHTSWTPQTPPLLAQNRTSWGDDQ 480

XM_031228099.1 AVIKVSDLPAKAEQTILLYAKVEKLAHMDYAPVGFINHTSWTPQTPPLLAQNRTSWDENQ 480
XM_031210721.1 TATKVSDLPAKAEQTILLYAKVEKLAHMDYAPVGFINHTSWTPQTPPLLAQNRTSWDENQ 480
XM_031192310.1 HUIB02 AATKVSDLPAKAEQTILLYAKVEKLAHMDYAPVGFINHTSWTPQTPPLLAQNRTSWDENQ 480
AATKVSDLPAKAEQTILLYAKVEKLAHMDYAPVGFINHTSWTPQTPPLLAQNRTSWDENQ 480
XM_018393180.1 AATKVSDLPAKAEQTILLYAKVEKLAHMDYAPVGFINHTSWTPQTPPLLAQNRTSWDENQ 480
:. ********************************************** **.::*
XM_018901654.1 LIPFIGISDRKPKRVDLIINNLDDGAHPFHLHGHSFYVLSSYRNPGRGSWGSYNPYTDEA 540

XM_031228099.1 LVPFIGISDSKPKRVDVVINNLDDGAHPFHLHGHSFYVLSSYRNPGRGSWGSYNPYTGEA 540
XM_031210721.1 LIPFIGISGSKPKRVDIVINNLDDGAHPFHLHGHSFYVLSSYRNPGRGSWGSYNPYTDEA 540
XM_031192310.1 HUIB02 LIPFIGISDSKPKRVDIVINNLDDGAHPFHLHGHSFYVLSSYRNPGRGSWGSYNPYTDEA 540
LIPFIGISDSKPKRVDIVINNLDDGAHPFHLHGHSFYVLSSYRNPGRGSWGSYNPYTDEA 540
XM_018393180.1 LIPFIGISDSKPKRVDIVINNLDGAHPFHLHGHSFYVLSSYRN:****** ******::**************************************.**
XM_018901654.1 PPNGLNLAFPVRKDTVSVPRRGHVVLALVADNPGIWALHCHMLVHMARGMAMGLHVGDIE 600

XM_031228099.1 PPNGLDLEFPVRKDTVSVPRRGHVVLALVADNPGIWALHCHMLVHMARGMAMGLHVGDID 600
XM_031210721.1 PPNGLNLEFPVRRDTVSVPRRGHVVLALVADNPGIWALHCHMLVHMARGMAMGLHVGDIE 600
XM_031192310.1 HUIB02 PPNGLNLEFLVRRDTVSVPRRGHVVLALVADNPGIWALHCHMLVHMARGMAMGLHVGDIK 600
PPNGLNLEFPVRRDTVSVPRRGHVVLALVADNPGIWALHCHTLVHMARGMAMGLRVGDIE 600
XM_018393180.1 PPNGLNLEFPVRRDTVSVPRRGHVVLALVADNPGIWALHCHMLVHMARGMAMGLHVGDIE 600
*****:* * **:**************************************** *:****.
XM_018901654.1 DPEHISSVDQRAAELCT 617

XM_031228099.1 DSEHVA------AELCT 611
XM_031210721.1 DPERVGSVDVRAAELCE 617
XM_031192310.1 HUIB02 DPEHVGSVDVRAAELCE 617
DPEHVGSVDVRAAELCE 617
XM_018393180.1 DPEHVGSVDVRAAELCE 617
* *::. ****
Figure 6. Comparative amino acid sequence of Folac3 gene of F. oxysporum HUIB02

strain with 5 other fungal strains. The differences in aa of the strains are represented by
a black background.
139
ATGTGGATTACAGACGCCTGCAAGGCTGTCGTCTTGTCCTTCGTGGCGGTATTCCACTACCCGT
CCGTCT
CAGACGAGGGGCCTCATCAAGATGTCATGACCTTCACCCACGGGCACTCCACACCAAAGATTG
ACTACCC
ATATTTCGACTCACCAAGTGGTCCTGACCAGGACGGCAAGGTCTTCACATGCAACTACACCGA
CCCAAA
GGCTGGAAGCCATGCACAACTGCAGATGATCGCTCGTGTTGGTTGAAAGGTCCGTATGGGCAA
AACTTCA
CTATTAACACAGACTATGAGAACTTCTGGCCAATTGGAATAACAAGAGAGGTACGTCGCGGCT
CATCAGC
ATTAGCTTGCTTTGCTTTCTGACTGACAACCAGTACCACCTCAACGTTACTGACCAATCCATCA
ATGGCG
ATGGTGTTGATAATCCATTCGGCAAGGTTTTCAACGGCAAGTATCCCGGCCCTTGGATCCAAG
CCCTG
GGGCGACCTCATCAAAGTCACGGTGCACAACCATCTCAAGTACAATGGTACAACTATCCACTG
GCACGGT
CTCCGACAACTGGAGACCTTTTGAGATGGATGGTGTCAACGGCGTGACTCAATGCCCCATTGCA
CCTGGGTG
ACAGTTATACCTACACCTTCCGTGCTGTTCAGTACGGTACTTCATGGTACCATAGCCATTATTC
TCTGCA
GTATGCTGATGGCTTGGCTGGCCCTATCACTATTTACGGGCCTTCTTCTGCGCCGTTCGATGAG
GGACGA
AATCCAATTCTTATCACCGATTGGAGTCATCGCAGTGCGTTCCAGTCTTGGCAGAGGGAGCTT
GTGCCGA
ATCCTACGAGACCTATGATGAATGGTGTTTTAATCAATGGCGTGGGGAACTTTGCTGGTAGTT
TCCCTCG
TGAGCGGTTCAACATGACTGTGACCAAGGTAGGCTACTTAAGCGCAGCAGCTGATAAGTCTGT
ACTGACA
ATCAATAGGGCAGGAAATATGTTCTTCGGGTGATCAACACCTCTGTGGCTACAACATGGATCT
TCAGTAT
CGACAATCACAACTTCACTGTTATGTCGACCGACTTTGTGCCGATCCATCCTTACTCAGTCTCT
CACATT
GTCCTCGGAATCGGTAAGCATATCTTCTCATTACAACCAATTGAAGACATTCTCACTGATGTCG
GTGAAC
AGGCCAGGGATATCACATTGTCCTGGATGCAAACCCCACCAACACCACAAAAATGCCCGCAA
ACCCCGAT
GGAAACTACTGGATTCGGACCGTCGGAGCAACTCGCTGCAAGGGCTTCGAGCCAGGCAATGA
GCCTGATG
AGCGACAAGGTATTCTACGGTACAATGCATTAAGCAAACTTGTTCCAACAACGTTCCGAGAGG
ACTATTC
CTTGGAATGTCGAGATGAAGCATATGATCGACTCAGCCCGGTTTACGAGTGGAATGTTGACCC
TGTCAAG
ATCAACTGTAAGTCACCGACGACCTTAATATTGTATGATTCCTCTAACTTTTGACTCAGACACC
AAGAGC
CAGTTCGACATTGGTCTAGCGAAGTTTGCACACAGGCCTGAGCTGATGGACAACTTCACCTGG
TGGGCCT
TTGGCGAGAATCCTCTCTGGTTGAACTTCTCCAACCCTACTATTCTGAATTCCAAGAACACCAC
ATGGAA
TCCCGACTATGCTGTGGATCTGGTGGTACCTGACTCGAAGAAAGATAAATGGGTCTACATCGC
TATCACA
140
GCCCCTCCGGCTCCCATTCAGACCAAACCCAACAGAGCTTTTGCTCCCGTTGCTCCATCCTGTAA
GCCAAC
CTCATCCCCTCCAAATAACTATCTCTAATGCGTAACAGCTCCATCTTCACGGTCACGACTTTGCC
CTTCTA
GCGCAAGGTACCAACTTCTCAGACCTGGACACAGGAAAGGTCAATCTCAAGTTCAACAACCCT
CCTCGTC
GAGATGTTGCGCTTCTTCCCGCCGGAGGATATCTCGTCGTTGCCTTCAAGGCCGACAACCCTG
GTTCTTG
GCTGTTCCACTGCCATATTGCGTGGCATGCGTCTAGTGGTCTGGCGATTCAGATCCTGGAGCA
ACAGACG
TTGCTGAATAGGATCTTGGATCAGTATCCTGAGAAGATGAAGGAGGTGAATAGAGTCTGTGAT
AACTGGA
ATAAGTGGTTCAAGAATGATACGAACCTTTGGAATGCCCATATCTTCCAGGATGACTCGGG
TATCTAA
Figure 7. Folac4 gene nucleotide sequence isolated from F. oxysporum strain HUIB02.
The sequence of the intron noncoding region is annotated with white letters on the background
black.
141
XP_018760174.1 MVAHDEEADALLEEFELREEVKVRDDRVLPLVCCTCGWRWIAALIVVALLSAIGVVVALH 60
XP_031078469.1 MVAHDEEADALLEEFELREEVKVRDARTPPSVCCTCGWRWIAALIVVALLSAIGVVVALY 60
XP_031059432.1 MVAHDEEADALLEEFELREEVKVRDDRTPPSVCRTCGWRWITALIVVALLSAIGRVREEADIVELVALLSAIGRVREEDRVELLVALLSAIGRVREEDRVELLVALLSA
XP_031031156.1 HUIB02 RDDRTPPSVCRTCGWRWITALIVVALLSAIGVVVVLH 60 MVAHDEEADALLEEFELREEVKVRDDRTPPSVCRTCGWRCMTALIVVALLSAIGVVVVLH
60 ************************* *. * ** ***** ::****************.*:
XP_018252396.1
XP_018760174.1 GLSSSQPSNDTTGHLGYRLHPQNHTSRPPTTLSFNWTITTGTRSPDGVEKQVYLVNDEFP 120

XP_031078469.1 GPSSSPPGNDTTGHLGYRLHPEDHAKRPPTTQYFNWTIAAGTKSPDGVEKRVYLVNNEFP 120
XP_031059432.1 GPLSNQLSDDATGHLGYRLHPQSHASRPPTTQSFNWTITAGTRSPDGVEKRVYLVNNEFP 120
XP_031031156.1 GLLSNQLSDDATGHLGYRLHPQSHASRPPTTQSFNWTITAGTRSPDGVEKRVYLVNNEFP 120
HUIB02 GPLSNQLSDDATSHLGYRLHPQSHASRPPTTQSFNWTITAGTRSPDGVEKRVYLVNNEFP 120
XP_018252396.1 GPLSNQLSDDATSHLGYRLHPQSHASRPPTTQSFNWTITAGTRSPDGVEKRVYLVNNEFP 120
* *. .:*:*.********:.*:.***** *****::**:*******:*****:** *
XP_018760174.1 GPLIEARSGDRVVIHVHNGLQDESVSLHWHGLRMKDQNNMDGAVGFTQCPIAPGRTFTYN 180

XP_031078469.1 GPLIEARSGDRIVIHVRNGLQDEGLSLHWHGLRMRYQNSMDGAVGFTQCPITPGTSFTYN 180
XP_031059432.1 GPLIEARSGDRLVIHVHNGVQDEGVSLHWHGLRMKYQNSMDGAVGFTQCPIAPGRSFTYN 180
XP_031031156.1 HUIB02 GPLIEARSGDRLVIHVHNGVQDEGVSLHWHGLRMKDQNSVDGAVGFTQCPIAPGRSFTYN 180
GPLIEARSGDRLVIHIHNGVQDEGVSLHWHGLRMKDQNSVDGAVGFTQCPIAPGRSFTYN 180
XP_018252396.1 GPLIEARSGDRLVIHVHNGVQDEGVSLHWHGLRMKDQNSVDGAVGFTQCPIAPGRSFTYN 180 ***********:***::**:***.:*********: **. :************:** :****
XP_018760174.1 FTIGAEEYGTFWWHSHSDVQRADGLWGGLVVHYPHEVDIPQEDYLVMIGDWFHQNQTEVL 240

XP_031078469.1 FTIGAEEQGTFWWHSHSDVQRADGLWGGLVQRADGLWGGLVVHSPDEVDVPPEDYLLMIGDWFHRNQTEVL 240
XP_031059432.1 FTIGAEEHGTFWWHSHSDSDVQRADGLWGGLIVHSPDEIDLWGGLIVQTSDEVLDEVDLWHQREEYLLEVGADLWHQREED
XP_031031156.1 HUIB02 EEHGTFWWHHPHSDVQRADGLWGGLIVHSPDEIDLQREEYLLMVGDWFHQNQTEVL 240
FTIGAEEHGTFWWHSHSDVQRADGLWGGLIVHSPDEIDLQREEYLLMVGDWFHQNQTEVL 240 ****.** ****** **************:** *.*:*: :* *:*:.****:******
XP_018252396.1
XP_018760174.1 RWYADASSRGNEPVPDSLLVNGKGRFNCSMVVPARPVACSQVQFSDLKPLMTSRSQNKVR 300

XP_031078469.1 GWYADASSRGNEPVPDSLLVNGRGRFNCSMAVPARPVVCSQVHLSDLKPLIMSGSLKKVR 300
XP_031059432.1 RWYADASSRGNEPVPDSLLLNGQGRFNCSMAVPARPVACSQVQFSDLKPLVMSRGQKKAR 300
XP_031031156.1 HUIB02 RWYADASSRGNEPVPDSMLVNGQGRFNCSMAVPARPIACSQVQFSDLKPLMMSRGQKKAR 300
RWYADASSRGNEPVPDSLLVNGQGRFNCSMAVPARPVACSQVQFSDLKPLMMSRSQKKAR 300
XP_018252396.1 RWYADASSRGNEPVPDSLLVNGQGRFNCSMAVPARPVACSQVQFSDLKPLMMSRGQKKAR 300
**************:*:**:*******.*****:.****::******: * . :*.*
XP_018760174.1 LRVVNTGSVAGLSLGVGRAILRPVRMDGGFAVKSEAAETVGTLYPGERVDLEVEWKRNHP 360

XP_031078469.1 IRVVNTGSVAGLSLRVGGAIIRPVRVDGGFAVKTEATETVGVLYPGERVDLEVEWKENYA 360
XP_031059432.1 LRVNAGSVAGLSLRVGGAIIRPVCVDGGFAVKAEATETVGILYPGERVDLEVEWKGNHA 360
XP_031031156.1 HUIB02 LRVVNTGSVAGLSLRVGGAIIRPVRVDGGFAVKAEATETVGILYPGERVDLEVEWKGNHA 360
LRVVNTGSVAGLSLRAGGAIIRPVRVDGGFAVKAEATETVGNLYPGERVDLEVEWKGNHA 360
XP_018252396.1 LRVVNTGSVAGLSLRVGGAIIRPVRVDGGFAVKAEATETVGILYPGERVDLEVEWKGNHA 360
:****:******** .* **:*** :********:**:**** ************ ** *:
XP_018760174.1 GDDWLTIYMDDENFGYPNLALNPTQSFPMFSSSTKGSFNEPVPQPLDEGAIQVLDSQNLK 420

XP_031078469.1 GDRWLTVYMDDENFGYPNPALHPTQSFPMFSSSAKGSFDEAVPQPLKQDEIQVLDSQNLK 420
XP_031059432.1 GDHRLTVYLDDENFGYPNPALNPTQSFPMFSSSTKGSSNEPVPQPLEQDEIQVLDSQNLK 420
XP_031031156.1 HUIB02 GDHRLTVYLDDENFGYPNPALNPTQSFPMFSSSTKEPSNEPVPQPLEQDEIQVLDSHNLK 420
GDHRLTVYLDDENFGYPNPALNPTQSFPMFSSSTKESSNEPVPQPLEQDEIQVLDSHNLK 420
XP_018252396.1 GDHRLTVYLDDENFGYSNPALNPTQSFPMFSSSTKESSNEPVPQALEQDEIQVLDSHNLK 420 ** **:*:******* * **:***********:* :* *** *. :. ******:***
XP_018760174.1 AAAKVSDLPPKAEQTILLYAKVEKLAHMDYAPVGFINHTSWTPQTPPLLAQNRTSWGDDQ 480

XP_031078469.1 AVIKVSDLPAKAEQTILLYAKVEKLAHMDYAPVGFINHTSWTPQTPPLLAQNRTSWDENQ 480
XP_031059432.1 TATKVSDLPAKAEQTILLYAKVEKLAHMDYAPVGFINHTSWTPQTPPLLAQNRTSWDENQ 480
XP_031031156.1 HUIB02 AATKVSDLPAKAEQTILLYAKVEKLAHMDYAPVGFINHTSWTPQTPPLLAQNRTSWDENQ 480
AATKVSDLPAKAEQTILLYAKVEKLAHMDYAPVGFINHTSWTPQTPPLLAQNRTSWDENQ 480
XP_018252396.1 AATKVSDLPAKAEQTILLYAKVEKLAHMDYAPVGFINHTSWTPQTPPLLAQNRTSWDENQ 480
:. ********************************************** **.::*
XP_018760174.1 LIPFIGISDRKPKRVDLIINNLDDGAHPFHLHGHSFYVLSSYRNPGRGSWGSYNPYTDEA 540

XP_031078469.1 LVPFIGISDSKPKRVDVVINNLDDGAHPFHLHGHSFYVLSSYRNPGRGSWGSYNPYTGEA 540
XP_031059432.1 LIPFIGISGSKPSKRVDIVINNLDDGAHPFHLHGHTDAHDFVIGHPSYRNPGYPFYLVSSYRNPGYPFYVLSSYRN
XP_031031156.1 HUIB02 LIPFIGISDSKPKRVDIVINNLDDGAHPFHLHGHSFYVLSSYRNPGRGSWGSYNPYTDEA 540
LIPFIGISDSKPKRVDIVINNLDDGAHPFHLHGHSFYVLSSYRNPGRGSWGSYNPYTDEA 540 *:******. ******::**************************************.**
XP_018252396.1
XP_018760174.1 PPNGLNLAFPVRKDTVSVPRRGHVVLALVADNPGIWALHCHMLVHMARGMAMGLHVGDIE 600

XP_031078469.1 PPNGLDLEFPVRKDTVSVPRRGHVVLALVADNPGIWALHCHMLVHMARGMAMGLHVGDID 600
XP_031059432.1 PPNGLNLEFPVRRDTVSVPRRGHVVLALVADNPGIWALHCHMLVHMARGMAMGLHVGDIE 600
XP_031031156.1 HUIB02 PPNGLNLEFLVRRDTVSVPRRGHVVLALVADNPGIWALHCHMLVHMARGMAMGLHVGDIK 600
PPNGLNLEFPVRRDTVSVPRRGHVVLALVADNPGIWALHCHTLVHMARGMAMGLRVGDIE 600
XP_018252396.1 PPNGLNLEFPVRRDTVSVPRRGHVVLALVADNPGIWALHCHMLVHMARGMAMGLHVGDIE 600
*****:* * **:**************************************** *:****.
XP_018760174.1 DPEHISSVDQRAAELC- 616

XP_031078469.1 DSEHVA------AELC- 610
XP_031059432.1 DPERVGSVDVRAAELCE 617
XP_031031156.1 HUIB02 DPEHVGSVDVRAAELCE 617
DPEHVGSVDVRAAELCE 617
XP_018252396.1 DPEHVGSVDVRAAELCE 617
* *::. ****
Figure 8. Comparative amino acid sequence of Folac4 gene of F. oxysporum HUIB02

strain with 5 other fungal strains. The differences in aa of the strains are represented by
a black background.
142
Appendix 8. Folac1 . gene sequencing peaks
143
Figure 1. Peak sequencing of the Folac1 gene in pGEM®T-Easy using

primer T7promoter
144
145
146
first
Figure 2. Peak sequencing of the Folac1 gene in pGEM®T-Easy using primer SP6Long
147
148
149
Figure 3. Peak sequencing of the Folac1 gene in pPICZÿA using primers AOX1-F
150
151
152
Figure 4. Peak sequencing of the Folac1 gene in pPICZÿA using primer AOX1-R
153
154

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DANG THI THANH HA

THESIS OF DOCTOR OF BIOTECHNOLOGY

RESEARCH TO CREATE REcombinant LACCASE

DANG THI THANH HA

RESEARCH FOR CREATION OF REcombinant LACCASE AND TESTING

THESIS OF DOCTOR OF BIOTECHNOLOGY

1. Assoc. Dr. Pham Thi Ngoc

AUTHORITY ................................................................................. ................................. .... iv

THANK YOU ............................................... ................................. ....v

LIST OF ACRONYMS............................................. ..................................... en LIST OF

TABLES. ................................. .............................. ix LIST OF

PICTURES ...... ................................. ................................................x

INTRODUCTION... ................................. ................................. ..............first

1. URGENTITY OF THE TOPIC .................................................................. ................................first

2. RESEARCH OBJECTIVES................................................................................. ............................2 3.

RESEARCH CONTENT... ................................. ................................2 4. NEW CONTRIBUTIONS OF THE

THESIS ....... .................................3 Chapter 1. TOTAL DOCUMENTARY

REVIEW ............................................................................................... ............4

1.1. LACCASE ....................................................................................... ................................. .........4

1.1.1. General introduction to laccases ..................................................................................... .................................4

1.1.2. Structure of the laccase .......................................................................... .................................4 1.1.3.

Catalytic mechanism of laccases .............................................................................. .................................6

1.1.4. Characterization of laccases ....................................................................................... ............................................8

1.1.5. Sources of laccase receipts ..................................................................................... ............11 1.1.6.

Effect of culture conditions on laccase biosynthesis .......14 1.1.7. Applications of

laccases ....................................................................................... ................................................15 1.2. Pichia

pastoris EXPRESSION SYSTEM ............................................................................ ............18 1.2.1. General

introduction to the P. pastoris system ..................................................................... ...........18 1.2.2. Advantages

TREATMENT MEASURES .......25 1.3.1. Industrial dyes ....................................................................................... ............25

1.3.2. Methods of treating industrial dyes ................................................................27 1.3.3. Color treatment

of dyes with laccase .................................................................................. .................28 Chapter 2. RESEARCH

SUBJECTS AND METHODS .................................................................. ......33

2.1. RESEARCH SUBJECTS .............................................. ............33 2.1.1.

Object ................................................ ................................. ......33 2.1.2. Sample

collection ................................................................................................. ................................. ..33 2.2.

RESEARCH METHODS.............................................. ............33

2.2.3. Determination of laccase enzyme activity ....................................................................... ............35

strains ....................................................... ............36

2.2.7. Isolation of laccase-coding genes ............38 2.2.8. cFolac1 expression in Pichia

pastoris ..................................................................... ............40 2.2.9. Investigating factors affecting the

expression of recombinant laccase ..........43 2.2.10. Purification of the recombinant

enzyme ....................................................................... ............44

2.2.11. SDS-PAGE electrophoresis .................................................................. .............45

ability of synthetic dyes to decolorize .......................................46 2.2.14. Statistical

processing ....................................................................................... ............................47 Chapter 3.

RESEARCH RESULTS ............................................................................ ............48 3.1. Isolation of

strains .......................................48 3.1. 2. Screening for laccase biosynthetic mold

strains ....................................................... 49 3.1 .3. Some morphological characteristics of colonies and

spore-forming stalks of fungal strains

isolation ................................................................................. ................................. .......50 3.1.4. Evaluation

of laccase-producing ability of isolated fungal strains .......................51 3.1.5. Investigating some

properties of natural laccases ..................................................................................... ........58

3.1.6. Identification of microorganisms ....................................................................................... .............60

laccase genes from the cDNA of F. oxysporum ................................................63

3.2.3. Sequencing the laccase

gene ....................................................................................... .................................71 3.3.

Expression of the LACCASE ENCORDING GENES IN Pichia pastoris..77 3.3.1. Cloning

expression vector pPICZÿA ................................................................................ ............77 3.3.3. Optimizatio

purification ....................................................................................... ................................................86

3.3.5. Characterization of recombinant enzymes ....................................................................... ................87

3.4. TESTING ABOUT RECOMMENDED LACCASE INDUSTRIAL

Dyestuffs .................................................................................. .................................................