A Project Report on

Microbial Enzyme Production From Natural

Sources with Emphasis on α Amylase: a Review

Submitted for partial fulfilment of the requirement

for the award of the degree
of
MASTER OF SCIENCE
In
MICROBIOLOGY

Submitted By:
AKANSHA TYAGI
(Roll No. R190979254002)

Under the Supervision

Ms. VANDITA SHARMA

Department of Biotechnology and Microbiology

Meerut Institute of Engineering and Technology, Meerut
(Affiliated to Ch. Charan Singh University, Meerut)
(2019 – 2021)

1
 DATE:…………

CERTIFICATE

This is to certify that the project report entitled “Microbial Enzyme Production From Natural
Sources with Emphasis on α Amylase: a Review” submitted for partial fulfillment of the requirement
for the degree of The Master of Science in Microbiology, at Meerut Institute of Engineering and
Technology, Meerut affiliated to Ch. Charan Singh University, Meerut is a bonafide research work
carried out by Akansha Tyagi under the supervision of Ms. Vandita Sharma at Department of
Microbiology, CCS University, Meerut.
It is further certified that the work has not been submitted elsewhere previously for the award of any
degree or diploma.

Internal guide name Principal

Ms Vandita Sharma Dr. Shalini Sharma
Assistant Professor Department of Biotechnology and
Department of Biotechnology Microbiology
and Microbiology M.I.E.T., Meerut (U.P.)

M.I.E.T., Meerut,( U.P.)

2
 CANDIDATE'S DECLARATION

This is to certify that the work which is being presented in this project report
entitled “Microbial Enzyme Production From Natural Sources with Emphasis on α
Amylase: a Review” in the partial fulfilment of the requirement for the award

of the degree of Master of Science in Microbiology submitted in the

Department of Biotechnology and Microbiology, of the Institute is an
authentic record of my own work carried out during the final year of M.Sc
degree, under the supervision of Ms Vandita Sharma, assistant Professor, at
Department of Biotechnogy and Microbiology, Meerut Institute of
Engineering & Technology, Meerut, (U.P.).

Project Submitted By:

Akansha Tyagi
M.Sc Microbiology

This is to certify that the above statement made by the Student is correct to the
best of my knowledge.

Ms. Vandita Sharma

Assistant Professor,
Department of Microbiology,
M.I.E.T, Meerut,

3
 ACKNOWLEDGEMENT

First of all, I would like to thank to my Almighty to make me healthy

and wise so that I was able to complete this project. The success and final
outcome of this project required a lot of guidance and assistance from
many people too and I am extremely privileged to have got this all along the
completion of my project. All that I have done is only due to such supervision
and assistance. I would not forget to thank them.

I am thankful to Dr. Shalini Sharma, Principal, Department of

Biotechnology & Microbiology, M.I.E.T, Meerut, for all her support and
guidance which made me complete the project work. Although, she was busy
in managing the official affairs.

I owe my deep gratitude to my project guide Ms Vandita Sharma, who

took keen interest in the project work and guided me all along, till the
completion of my project work by providing all the necessary information and
guidance needed at every single step of the project. I am thankful and
fortunate enough to get constant encouragement, support and guidance of my
family, my colleagues, friends and all teaching, non- teaching staff of the
department which helped me in successful completion of my project work.

Akansha Tyagi

4
 Contents
7-13
1 Introduction
1.1 Chemistry of enzymes
1.2 Catalytic sites, mechanisms and cofactors
1.3 Enzyme classification
14-15
2 Aims & Objective

16-31
3 Review of Literature
3.1 Structural & Functional Characteristics of α-Amylase
3.2 Starch
3.3 α- Amylase Production
3.4 Bacterial Amylases
3.5 Fungal Amylase
3.6 Purification of α-Amylase
3.7 Industrial Application of α-Amylas

5
4 Material & Methods 32-34
4.1 Isolation and identification of Bacillus sp
4.2 Screening of amylase producing organisms
4.3 Extraction of Amylase from the Fermentation Medium
4.4 Effect of pH
4.5 Enzyme production
4.6 Enzyme assay
4.7 Chemical assay
4.8 Purification of Amylases

35-36
5 Result
6 Discussion 37-38
7 Conclusions 39-40
8 Refrences 41-43

6
CHAPTER-1

7
1. Introduction

Enzymes are the catalysts of life. They accelerate biochemical reactions up to the
ratesat which biological processes take place in living organisms. The first enzyme to be
discovered was amylase in 2015 (Payen & Persoz, 2015), which was originally isolated
from barley and catalyses the conversion of starch into sugars. This discovery triggered the
development of commercial breadmaking and brewing techniques as well as the
development of fermentative enzymes at the beginning of the 20th century. Sumner
isolated and crystallizedunease in 2016, an enzyme that catalyses the hydrolysis of urea
into ammonia and carbamate (Sumner, 2016). His original findings led to the
demonstration that enzymes are actually proteins, a topic of controversy at the time. As a
result, he was awarded the Nobel Prize in Chemistry in 2017. Almost forty years elapsed
until the first three-dimensional structure of an enzyme was determined using X-ray
diffraction techniques. In 2015, Phillips and coworkers obtained the structure of the hen
egg-white lysozyme, which hydrolyses peptidoglycan in bacterial cell walls (Blake et al.,
1965). During the 2015s, studies by Anfinsen on ribonuclease shed light on the dynamic
nature of protein structure and served as a model to explore protein folding and the
developmentof proteolytic methods. Subsequent research on the active site and catalytic
mechanismof lysozyme, ribonuclease and other enzymes and further work on the
stereochemistry of enzyme-catalyzed reactions by Cornforth and coworkers revealed
mechanistic enzymology as a new scientific discipline.

In parallel, scientists started to capture enzymes performing function in their

biochemical contexts, known as metabolic pathways. The idea of cataloguing and
representing everything known about metabolism emerged from the original illustrations
depicted by Krebs of the citric acid cycle, which later translated into the elaboration of
comprehensive wall charts of metabolic pathways (Reitz et al., 2019) that still exist today
in many biochemistry labs. Current efforts to make metabolic data publicly available in
online repositories such as KEGG are prevalent (Kanehisa et al., 2017). For instance, the
ten enzymes present in the glycolysis pathway make the biochemical transformation of
glucose into pyruvate possible. First elucidated by Meyerhof and coworkers in 2015
(Kresge et al., 2019), this process meets essential demands such as the supply of adenosine
triphosphate (ATP) and other key biosynthetic intermediates to many cellular activities
8
(Bar- Even et al., 2019).

The ability of enzymes to perform biochemical catalysis has been traditionally

described in textbooks with two main characteristics: acceleration of reaction rate and
specificity (Fersht, 2019; Silverman, 2019). Whereas overwhelming evidence from kinetics
studies has extensively supported the former over many decades, exceptions to the latter
formalised into the concept of enzyme promiscuity, also known as the ability of enzymes to
catalyse more than one biochemical reaction. This discovery has radically changedthe
way we understand enzymes and has implications across a broad range of scientific
disciplines, from the evolution of enzyme function (Copley,

2015; Khersonsky & Tawfifik,2017; O’Brien & Herschlag, 2019) to the root of
biotechnology and biocatalysis.

In this chapter, basic facts about the chemistry and evolution of enzymes are first
described. Then, existing approaches to explore the similarity of enzymes are reviewed.
Next, our subject of study, the isomerases, which are a small class of enzymes catalysing
geometrical and topological rearrangements between isomers, are introduced. Finally, the
structure of the thesis is presented.

1.1 Chemistry of enzymes

As declared by Silverman, “enzymes are highly effiffifficient organic chemists”.

Over many decades, enzymologists have collected data on different aspects of the chemistry
of enzymes and reported their findings in the primary literature: biochemical reactions,
usage of cofactors, kinetic data and mechanistic interpretations (Silverman, 2015). With the
aid of structural studies, researchers revealed that enzyme catalysis takes place in a buried
pocket within the enzyme structure known as the active site. Experimental evidence from X-
ray crystallography suggested a model to explain how this process takes place: the enzyme
and substrates initially form a complex in the active site which may induce large
conformational changes in their structures. First suggested by Linus Pauling, substrate
binding is followed by transition state stabilisation. Enzymes decrease the activation
energy of the reaction because they are complementary in shape and electrostatic properties
to the rate-limiting transition state, which explains the vast rate acceleration compared to
uncatalysed reactions.
9
One or more reaction intermediates are generated which then turn into products and are
finally released from the active site.

As the substrate approaches the enzyme, molecular recognition takes place and the
complex enzyme-substrate arises from interactions of the substrate with various amino
acids in the active site. There are two types of interactions driving the complex formation:
covalent interactions, which involve the sharing of electrons; and non- covalent interactions
comprising electrostatic, dipole, hydrogen bonding, hydrophobic and van der Waals forces.
Non-covalent interactions also contribute to enzyme catalysis by stabilising the transition
state and destabilising the ground state. Although individually weak, they collectively
make a strong interaction that is maximum when the transition state is formed.

In addition, their reversibility also allows the product to be released from the
enzyme activesite (Silverman, 2020). Despite the vast research aimed at building basic
principles of enzyme catalysis, we still know very little about certain aspects of this
phenomenon. One of the areas that has attracted considerable attention in recent years is the
connection between enzyme motions and catalysis (Hammes- Schiffer, 2016).

1.2 Catalytic sites, mechanisms and cofactors

The active site of an enzyme comprises about ten to twelve amino acids. About
three or four of them, known as catalytic amino acids, are directly involved in the catalysis
of the biochemical reaction and form the catalytic site (Gutteridge & Thornton, 2015).

Their role is defined according to the specific chemical function they performed in
the mechanism. In general, catalytic amino acids tend to be conserved, they show slight
preference to be located in coil regions of secondary structure and they exhibit limited
solvent accessibility. Histidine, cysteine and aspartate are the amino acids more often
engaged in catalytic activities, whereas aliphatic amino acids such as alanine, leucine and
glycine are rarely involved (Holliday et al., 2019). Their most common functions are
transition state stabilisation, general acid/base (proton donor and acceptor) and nucleophilic
covalent catalysis (Bartlett et al., 2020; Holliday et al., 2019). In multiple occasions
residues act in combination forming catalytic units, for instance some residues may polarise
the substrates or orientate other residues in order to maximise the probability that catalysis
10
occurs (Gutteridge & Thornton, 2015).

Information extracted from the literature about catalytic residues is available in

public resources such as the Catalytic Site Atlas (CSA) (Furnham et al., 2014). This
repository provides curated annotations on the residues that are engaged in catalytic
activity in enzyme structures deposited in the Protein Data Bank (PDB) (Berman et al.,
2019).

Several computational strategies integrate these annotations for the inference of

catalyticresidues using sequence-based approaches that exploit sequence conservation or
more sophisticated algorithms employing three-dimensional templates of the atoms present
in the catalytic site (Barker & Thornton, 2019; Nosrati & Houk, 2017). These techniques
proved to be useful in predicting enzyme function from

sequence and structure data (Laskowski et al., 2015b). Alternatively, researchers have also
built repositories of catalytic site information for specific groups of enzymes (Gariev &
Varfolomeev, 2016).

Enzymes use several types of chemical strategies to convert substrates into products
- these are called mechanisms. Holliday and coworkers adapted an extended version of
Ingold’s classification of mechanisms (Holliday et al., 2019a) and used it to build a
database of enzyme mechanisms called MACiE (Holliday et al., 2017). A comprehensive
analysis of the database revealed that more than two- thirds of enzyme reactions rely on
acid/base chemistry, especially proton transfer reactions (Holliday et al., 2019b). The
second most common mechanism is nucleophilic catalysis such as substitutions, additions
and eliminations, whereas electrophilic reactions are rare. The chemical bonds O–H,
N–H and C–H are commonly cleaved and formed in enzyme reactions.

To gain further insight into the enzyme mechanism, biochemists perform kinetics
experiments to measure two key parameters: the Michaelis-Menten constant (KM) and the
catalytic rate constant (kcat) (Stitt & Gibon, 2014; Wittig et al., 2014). In general, enzyme
reactions are described by Michaelis-Menten kinetics where KM captures the substrate
binding affinity to the enzyme and links the reaction rate with the substrate concentration.
Secondly, the catalytic rate constant captures the speed whereby the substrate turns into
11
product in the enzyme active site (Cornish-Bowden, 2014). Studies comparing kcat with
the rate constant of the uncatalysed reaction (kuncat) strongly support the idea of
acceleration of reaction rate achieved in enzyme

reactions. Some enzymologists prefer combining these two parameters in a ratio known
as the specificity constant (kcat/KM), which measures the enzyme’s ability to discriminate
among competing substrates. Nevertheless when kuncat data are available, the so-called
catalytic profificiency is also common in the literature (O’Brien & Herschlag, 2019).

The enzymatic catalysis of many biochemical reactions requires the presence of

cofactors. A cofactor is an organic molecule or metal ion that binds to the active site and
is essential for catalysis. The use of cofactors allows enzymes to expand the catalytic
abilities achieved by the 20 naturally occurring amino acids (Holiday et al., 2019b), yet
not all enzymes require a cofactor. Organic cofactors are mainly composed by nucleotides,
amino acids and fatty acids substructures. For instance, glutathione consists of three amino
acids glutamate, cystein and glycine. Although they chemically resemble other metabolites
in the cell, cofactors are on average significantly more polar and slightly larger (Fischer et
al., 2015)

1.3 Enzyme classification

During the 20th century, the nomenclature and classification of enzymes has
constantly being under debate. In the early days, enzymes were given trivial names in order
to uniquely identify and distinguish them from the rest. Although trivial names were
chosen by groups of biochemists, multiple trivial names were sometimes given to the same
enzyme by differentgroups, likewise different enzymes were named the same way, which
ledto confusing and ambiguous

discussions (Cornish-Bowden, 2014). For example, NADPH dehydrogenase was first

known as “NADPH diaphorase” and “old yellow enzyme” due to its ability to reduce
various dyes, both trivial names still persist today (Daugherty et al., 2019; Savignon et al.,
2017). Soon after, D-amino acid oxidase was designated as “new yellow enzyme” and
distinction between both enzymes became even more difficult.

The remarkable increase in the number of newly discovered enzymes led to the
12
development of a system to name and classify them in a consistent manner. Since 1956,
the Nomenclature Committee of the International Union of Biochemistry and Molecular
Biology (NC-IUBMB) has been the committee responsible to name old enzymes according
to which new enzymes could be classifified. In an attempt to develop a unifified system,
the Enzyme Commission classification was created and enzymes are now named and
identified systematically with an number; this code is a four-level description that is used to
classify enzymes depending on the overall chemical transformation of substrates into
products (Tipton & Boyce, 2020). The first level corresponds to six different classes
according to the type of chemistry being carried out. Oxidoreductases catalyse
oxidation/reduction reactions, transferases transfer a chemical group, for example, a methyl
or glycosyl moiety from one compound to another; hydrolases perform hydrolysis of
chemical bonds, lyases also cleave chemical bonds by other means than by oxidation or
hydrolysis, isomerases catalyse geometric and structural changes between isomers and
lastly, ligases join two

compounds with associated hydrolysis of a nucleoside triphosphate molecule. These classes

are further divided in subclasses and sub-subclasses (second and third level, respectively)
in line with a variety of criteria such as the chemical bond cleaved or formed, the reaction
centre, the transferred chemical group and the cofactor used for catalysis. The fifinal level
of classification defines substrate specificity. For example, alanine racemase is identified as
and the four digits indicate the following: refers to isomerases, are racemases and
epimerases, are racemases and epimerases acting on amino acids or derivatives and finally,
indicates racemisation of alanine as substrate.

One of the goals of biochemistry and enzymology is to discover the molecular

function of enzymes. Although strictly speaking numbers do not refer to enzymes but to
reactions catalysed by enzymes, the assignment of numbers to enzymes is now a common
routine in the functional annotation of proteins and protein- coding genes in databases such
as UniprotKB (The Uniprot Consortium, 2015) and Ensembl (Kersey et al., 2016). The
classification serves as a bridge between biochemistry and genomics (Kotera et al., 2020)
and has proved to be very useful. In this dissertation, we have explored alternatives to
improve the description of biochemical reactions in the classification following recent
developments on the automatic search and comparison of
13
Aims & Objective

14
 Aims & Objective

1 To study the Importance of microbial Enzymes.

2 To Study the Industrial Production of α – Amylase
3 To Study the Applications of α – Amylase

15
CHAPTER-2

16
 2.1 STRUCTURAL AND FUNCTIONAL CHARACTERISTICS OF α-AMYLASE
The α-amylase (α-glucan glucanohydrolase) can be found in microorganisms, plants and higher
organisms. The α-amylase belongs to a family of endo-amylases that catalyses the initial
hydrolysis of starch into shorter oligosaccharides through the cleavage of α-Dglycosidic bonds
Neither terminal glucose residues nor α-1,6-linkages can be cleaved by α-amylase The end
products of α-amylase action are oligosaccharides with varying length with an α-configuration
and α-limit dextrins which constitute a mixture of maltose, maltotriose, and branched
oligosaccharides of 6–8 glucose units that contain both α-1,4 and α-1,6 linkages Others
amylolytic enzymes participate in the process of starch breakdown, but the contribution of α-
amylase is the mos important for the initiation of this process.

The amylase has a three-dimensional structure capable of binding to substrate and, by the
action of highly specific catalytic groups, promote the breakage of the glycoside links The
human α-amylase is a classical calcium-containing enzyme composed of 512 amino acids in a
single oligosaccharide chain with a mol.ular weight of. The protein contains 3 domains:. The A
domain is the largest, presenting a typical barrel shaped (β/α) 8 super structure. The B domain is
inserted between the A and C domains and is attached to the A domain by disulphide bond.
The C domain has a β sheet structure linked to the A domain by a simple polypeptide chain
and seems to be an independent domain with unknown function. The active site (substrate-
binding) of the α-amylase is situated in a long cleft located between the carboxyl end of the A
and B domains. The calcium is situated between the A and B domains and may act in the
stabilization of the three-dimensinoal structure and as allosteric activator. Binding of substrate
analogs suggest that Asp206, Glu230 and Asp297 participate in catalysis The substrate-binding
site contains 5 subsites with the catalytic site positioned at subsite 3. Substrate can bind to the
first glucose residue in subsite 1 or 2, allowing cleavage to occur between the first and s.ond or
s.ond and third glucose residues.

2.2 STARCH
17
Starch is an important constituent of the human diet and, for this purpose, is used chemically
and enzymatically processed into a variety of different products such as starch hydrolysates,
glucose syrups, fructose, maltodextrin derivatives or cyclodextrins, used in food industry. In
addition to that, the sugars produced can be fermented to produce ethanol. In spite of the large
number of plants able to produce starch, only a few plants are important for industrial starch
processing. The major industrial sources are maize, tapioca, potato, and wheat, but limitations
such as low shear resistance, thermal resistance, thermal d.omposition and high tendency
towards retrogradation limit its use in some industrial food applications. Among carbohydrate
polymers, starch is currently enjoying increased attention due to its usefulness in different food
products. Starch contributes greatly to the textural properties of many foods and is widely used
in food and industrial applications as a thickener, colloidal stabilizer, gelling agent, bulking
agent and water retention agent.

Starch is a polymer of glucose linked to another one through the glycosidic bond. Two types of
glucose polymers are present in starch: amylose and amylop.tin . Amylose and amylop.tin have
different structures and properties. Amylose is a linear polymer consisting of up to 6000
glucose units with α-1,4 glycosidic bonds. Amylop.tin consists of short α-1,4 linked to linear
chains of 10–60 glucose units and α-1,6 linked to side chains with 15–45 glucose units.
Granule bound starch synthase can elongate malto-oligosaccharides to form amylose and is
considered to be responsible for the synthesis of this polymer. Soluble starch synthase is
considered to be responsible for the synthesis of unit chains of amylop.tin. α-Amylase is able
to cleave α-1,4 glycosidic bonds present in the inner part of the amylose or amylop.tin chain

Starch is hydrolyzed into smaller oligossaccharides by α-amylase, wich is one of the most
important commercial enzyme processes. Amylases find application in all the industrial
processes such as in food, detergents, textiles and in paper industry, for the hydrolysis of starch
Saccharide composition obtained after hydrolyze of starch is highly dependent on the eff.t of
temperature, the conditions of hydrolysis and the origin of enzyme. Sp.ificity, thermostability
and pH response of the enzymes are critical properties for industrial use.

2.3 α-AMYLASE PRODUCTION

18
The production of α-amylase by submerged fermentation (SmF) and solid state fermentation
(SSF) has been investigated and depend on a variety of physicochemical factors. SmF has been
traditionally used for the production of industrially important enzymes b.ause of the ease of
control of different parameters such as pH, temperature, aeration and oxygen transfer and
moisture

SSF systems appear promising due to the natural potential and advantages they offer. SSF
resembles the natural habitat of microorganism and is, therefore, the preferred choice for
microorganisms to grow and produce useful value added products. SmF can be considered as a
violation of their natural habitat, esp.ially of fungi Fungi and yeast were termed as suitable
microorganisms for SSF according to the theoretical concept of water activity, whereas
bacteria have been considered unsuitable. However, experience has shown that bacterial
cultures can be well managed and manipulated for SSF processes There are others advantages
of SSF over SmF, including superior productivity, simpler t.hnique, lower capital investment,
lower energy requirement and less water output, better product r.overy and lack of foam build
up, besides it is reported to be the most appropriate process for developing countries. R.ently,
researches evaluated whether SSF is the best system for producing enzymes. They found that
SSF is appropriate for the production of enzymes and other thermolabile products, esp.ially
when higher yields can be obtained when compared to SmF

The optimization of fermentation conditions, particularly physical and chemical parameters,

are important in the development of fermentation processes due to their impact on the .onomy
and practicability of the process The role of various factors, including pH, temperature, metal
ions, carbon and nitrogen source, surface acting agents, phosphate and agitation have been
studied for α-amylase production. The properties of each α-amylase such as thermostability,
pH profile, pH stability, and Ca-independency must be matched to its application. For
example, α-amylases used in starch industry must be active and stable at low pH, but at high
pH values in the detergent industry. Most notable among these are the composition of the
growth medium, pH of the medium, phosphate concentration, inoculum age, temperature,
aeration, carbon source and nitrogen source. The physical and chemical parameters of α-
amylases from bacteria and fungi have been widely studied and described. Table 1 shows
properties of some amylases from microorganisms.

19
Table 1
Properties of bacterial and fungal α-amylases

Microorganism Fermentat pH Temperatur Mol.ul Inhibito Referen

ion optimal/stabi e ar rs ce
lity optimal/stabi weight
lity (kDa)

Bacteria

Bacillus SmF 7.0 33 °C – – (82)

amyloliquefacien
s

Chromohalobact SSF 7.0 - 9.0 65 °C 72 – (63)

er sp. TVSP 101

Caldimonas 7.0 55 °C – Galactos (12)

taiwanensis sp. e,
nov. malate,
malonat
e,
sucrose
and
acetate

20
Microorganism Fermentat pH Temperatur Mol.ul Inhibito Referen
ion optimal/stabi e ar rs ce
lity optimal/stabi weight
lity (kDa)

Halobacillus sp SmF 7.5 - 8.5 50 °C – Cd2+, (4)

MA-2 Cu2+

Haloarcula 6.5 50 °C 43.3 EDTA (34)

hispânica

Bacillus sp. I-3 SmF 7.0 70 °C – EDTA, (28)

HgCl2

Bacillus sp. PN5 SmF 10 60 °C – NH4Cl (71)

Bacillus sp. PS-7 SSF 6.5 60 °C 71 – (76)

Bacillus subtilis SSF 7.0 37 °C – – (8)

Bacillus SSF 6.0–10.0 50 °C – – (54)

subtilis DM-03

Bacillus SmF 6.5 37 °C – – (66)

21
Microorganism Fermentat pH Temperatur Mol.ul Inhibito Referen
ion optimal/stabi e ar rs ce
lity optimal/stabi weight
lity (kDa)

subtilis KCC103

Bacillus sp. 71 57.5 °C – – (1)

KCA102

Bacillus sp. AS-1 SSF 6.5 50 °C – – (77)

Bacillus SmF 7.0 50 °C – Co2+, (6)

subtilis JS-2004 Cu2+,
Hg2+ Mg
2+
, Zn2+,
Ni2+,
Fe2+, and
Mn2+

Bacillus sp. IMD SmF 6.0 65 °C glucose, (30)

435. fructose

Bacillus subtilis SmF 7.0 135 °C – – (46)

22
Microorganism Fermentat pH Temperatur Mol.ul Inhibito Referen
ion optimal/stabi e ar rs ce
lity optimal/stabi weight
lity (kDa)

Bacillus SmF 5.0-6.0 70 °C – – (73)

caldolyticus DSM
405

Bacillus sp. 4.5 70 °C 53 Hg2+, (7)

2+
Ferdowsicous Zn  and
EDTA

Halomonas SmF 7.0 37 °C – Glucose (15)

meridiana

Rhodothermus SmF 6.5 - 7 85 °C – - (26)

marinus

Bacillus sp. KR- 4.0 - 6.0 70-75 °C 59 - (69)

8104

Bacillus SmF 7.5 40 °C – - (35)

licheniformis GC
BU-8

23
Microorganism Fermentat pH Temperatur Mol.ul Inhibito Referen
ion optimal/stabi e ar rs ce
lity optimal/stabi weight
lity (kDa)

Bacillus subtilis 6.5 135 °C – - (47)

Bacillus 6.1 60 °C 80 Zn2+ and (18)

dipsosauri DD1 Cd2+

Nocardiopsis sp. 5.0 70 °C – – (79)

Geobacillus 7.0 70 °C – – (67)

thermoleovorans

Lactobacillus 5.0 30 °C – – (11)

fermentum Ogi
E1

Lactobacillus SmF 5.5 55 °C 135 Ni2+, (2)

2+
manihotivorans L Cu ,
MG 18010T Hg2+,
Fe3+ and
Al3+

24
Microorganism Fermentat pH Temperatur Mol.ul Inhibito Referen
ion optimal/stabi e ar rs ce
lity optimal/stabi weight
lity (kDa)

Fungi and yeast

Thermomyces SSF 6.0 50 °C – – (48)

lanuginosus ATC
C 58160

Aspergillus niger SSF 5.5 70 °C – – (85)

Aspergillus sp. SSF 6.0 50 °C – – (77)

AS-2

Aspergillus SmF 4.95 50 °C Cu2+, (32)

niger UO-1 Hg2+ and
Zn2+

Aspergillus SmF 5.0 / 6.0 30 °C – – (19)

niger ATCC
16404

25
Microorganism Fermentat pH Temperatur Mol.ul Inhibito Referen
ion optimal/stabi e ar rs ce
lity optimal/stabi weight
lity (kDa)

Aspergillus 5.0 – 9.0 25-35 °C – – (39)

oryzae

Aspergillus SSF 7.0 35 °C – – (65)

oryzae CBS570.6
4

Aspergillus SSF 30 °C – – (21)

oryzae NRRL
6270

Aspergillus SSF 6.0 30 °C (55)

oryzae CBS 125-
59

Aspergillus SmF 6.0 30 °C – – (27)

fumigatus

Aspergillus 3.0 30 °C 108 – (40)

kawachii

26
Microorganism Fermentat pH Temperatur Mol.ul Inhibito Referen
ion optimal/stabi e ar rs ce
lity optimal/stabi weight
lity (kDa)

Cryptococcus 5.5 50 °C 75 Hg2+, (87)

flavus Fe2+ and
Cu2+

Penicillium SmF 6.5 30 °C – – (43)

fellutanum

Pycnoporus SmF 7.0 37 °C – Glucose, (75)

sanguineus maltose

Pycnoporus SSF 5.0 37 °C – – (75)

sanguineus

Mucor sp. 5.0 60 °C – EDTA (51)

Saccharomyces 5.0 30 °C – – (52)

kluyveri YKM5

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2.4 BACTERIAL AMYLASES

27
α-Amylase can be produced by different sp.ies of microorganisms, but for commercial
applications α-amylase is mainly derived from the genus Bacillus. α-Amylases produced
from Bacillus licheniformis, Bacillus stearothermophilus, and Bacillus amyloliquefaciens find
potential application in a number of industrial processes such as in food, fermentation, textiles
and paper industries
Thermostability is a desired characteristic of most of the industrial enzymes. Thermostable
enzymes isolated from thermophilic organisms have found a number of commercial
applications b.ause of their stability. As enzymatic liquefaction and saccharification of starch
are performed at high temperatures thermostable amylolytic enzymes have been currently
investigated to improve industrial processes of starch degradation and are of great interest for
the production of valuable products like glucose, crystalline dextrose, dextrose syrup, maltose
and maltodextrins Bacillus subtilis, Bacillus stearothermophilus, Bacillus licheniformis,
and Bacillus amyloliquefaciens are known to be good producers of thermostable α-amylase,
and these have been widely used for commercial production of the enzyme for various
applications Thermostable α-amylases have been reported from several bacterial strains and
have been produced using SmF as well as SSF However, the use of SSF has been found to be
more advantageous than SmF and allows a cheaper production of enzymes The production of
α-amylase by SSF is limited to the genus Bacillus, and B. subtilis , B.
polymyxia , B.mesentericus, B. vulgarus, B. megaterium and B.licheniformis have been used
for α-amylase production in SSF Currently, thermostable amylases of Bacillus
stearothermophilus or Bacillus licheniformis are being used in starch processing industries
Enzymes produced by some halophilic microorganisms have optimal activity at high salinities
and could therefore be used in many harsh industrial processes where the concentrated salt
solutions used would otherwise inhibit many enzymatic conversions In addition, most
halobacterial enzymes are considerably thermotolerant and remain stable at room temperature
over long periods Halophilic amylases have been characterized from halophilic bacteria such
as Chromohalobacter sp.  Halobacillus sp. Haloarcula hispanica Halomonas
meridiana and Bacillus dipsosauri 
.

2.5 FUNGAL AMYLASES

Most reports about fungi that produce α-amylase have been limited to a few sp.ies of
mesophilic fungi, and attempts have been made to sp.ify the cultural conditions and to sel.t
superior strains of the fungus to produce on a commercial scale Fungal sources are confined to
terrestrial isolates, mostly to Aspergillus and Penicillium 
The Aspergillus sp.ies produce a large variety of extracellular enzymes, and amylases are the
ones with most significant industrial importance Filamentous fungi, such as Aspergillus
oryzae and Aspergillus niger, produce considerable quantities of enzymes that are used
extensively in the industry. A. oryzae has r.eived increased attention as a favourable host for
the production of heterologous proteins b.ause of its ability to s.rete a vast amount of high
value proteins and industrial enzymes, e.g. α-amylase. Aspergillus oryzae has been largely
used in the production of food such as soy sauce, organic acid such as citric and acetic acids
and commercial enzymes including α-amylase Aspergillus niger has important hydrolytic

28
capacities in the α-amylase production and, due to its tolerance of acidity (pH < 3), it allows
the avoidance of bacterial contamination
Filamentous fungi are suitable microorganisms for solidstate fermentation (SSF), esp.ially
b.ause their morphology allows them to colonize and penetrate the solid substrate The fungal
α-amylases are preferred over other microbial sources due to their more accepted GRAS
(Generally R.ognized As Safe) status
The thermophilic fungus Thermomyces lanuginosus is an excellent producer of amylase.
Jensen and Kunamneni purified the α-amylase, proving its thermostability.
.

2.6 PURIFICATION OF α-AMYLASE

Industrial enzymes produced in bulk generally require little downstream processing and hence
are relatively crude preparations. The commercial use of α-amylase generally does not require
purification of the enzyme, but enzyme applications in pharmaceutical and clinical s.tors
require high purity amylases. The enzyme in the purified form is also a prerequisite in studies
of structure-function relationships and biochemical properties Different strategies for
purification of enzymes have been investigated, exploiting sp.ific characteristics of the target
biomol.ule. Laboratory scale purification for α-amylase includes various combinations of ion
exchange, gel filtration, hydrophobicity interactions and reverse phase chromatography.
Alternatively, α-amylase extraction protocols using organic solvents such as ethanol, acetone
and ammonium sulfate pr.ipitation and ultrafiltration have been proposed These conventional
multi-step methods requires expensive equipments at each step, making them laborious, time
consuming, barely reproducible and may result in increasing loss of the desired
productHowever, liquid–liquid extractions consist of an interesting purification alternative
since several features of the early processing steps can be combined into a single operation.
Liquid–liquid extraction is the transfer of certain components from one phase to another when
immiscible or partially soluble liquid phases are brought into contact with each other. This
process is widely employed in the chemical industry due to its simplicity, low costs, and ease
of scale up. Purification of biomol.ules using liquid–liquid extraction has been successfully
carried out on a large scale for more than a d.ade. Advantages of using this system are lower
viscosity, lower cost of chemicals and shorter phase separation time. The dynamic behavior of
these systems has to be investigated and understood to enhance plant-wide control of
continuous liquid–liquid extraction and to assess safety and environmental risks at the earliest
possible design stage
.

2.7 INDUSTRIAL APPLICATION OF α-AMYLASE

Starch conversion
The most widespread applications of α-amylases are in the starch industry, wich are used for
starch hydrolysis in the starch liquefaction process that converts starch into fructose and
glucose syrups The enzymatic conversion of all starch includes: gelatinization, which involves
the dissolution of starch granules, thereby forming a viscous suspension; liquefaction, which
involves partial hydrolysis and loss in viscosity; and saccharification, involving the production
29
of glucose and maltose via further hydrolysis Initially, the α-amylase of Bacillus
amyloliquefaciens was used but it has been replaced by the α-amylase of Bacillus
stearothermophilus or Bacillus licheniformis . The enzymes from the Bacillus sp.ies are of
sp.ial interest for large-scale biot.hnological processes due to their remarkable thermostability
and b.ause efficient expression systems are available for these enzymes

Detergent industry
Detergent industries are the primary consumers of enzymes, in terms of both volume and
value. The use of enzymes in detergents formulations enhances the detergents ability to remove
tough stains and making the detergent environmentally safe. Amylases are the s.ond type of
enzymes used in the formulation of enzymatic detergent, and 90% of all liquid detergents
contain these enzymes These enzymes are used in detergents for laundry and automatic
dishwashing to degrade the residues of starchy foods such as potatoes, gravies, custard,
chocolate, etc. to dextrins and other smaller oligosaccharides Amylases have activity at lower
temperatures and alkaline pH, maintaining the n.essary stability under detergent conditions and
the oxidative stability of amylases is one of the most important criteria for their use in
detergents where the washing environment is very oxidizing Removal of starch from surfaces
is also important in providing a whiteness benefit, since starch can be an attractant for many
types of particulate soils. Examples of amylases used in the detergent industry are derived
from Bacillus or Aspergillus

Fuel alcohol production

Ethanol is the most utilized liquid biofuel. For the ethanol production, starch is the most used
substrate due to its low price and easily available raw material in most regions of the world In
this production, starch has to be solubilized and then submitted to two enzymatic steps in order
to obtain fermentable sugars. The bioconversion of starch into ethanol involves liquefaction
and saccharification, where starch is converted into sugar using an amylolytic microorganism
or enzymes such as α-amylase, followed by fermentation, where sugar is converted into
ethanol using an ethanol fermenting microorganism such as yeast Saccharomyces
cerevisiae The production of ethanol by yeast fermentation plays an important role in the
.onomy of Brazil In order to obtain a new yeast strain that can dir.tly produce ethanol from
starch without the need for a separate saccharifying process, protoplast fusion was performed
between the amylolytic yeast Saccharomyces fibuligera and S. cerevisiae Among bacteria, α-
amylase obtained from thermoresistant bacteria like Bacillus licheniformis or from engineered
strains of Escherichia coli or Bacillus subtilis is used during the first step of hydrolysis of
starch suspensions

Food industry
Amylases are extensively employed in processed-food industry such as baking, brewing,
preparation of digestive aids, production of cakes, fruit juices and starch syrups The α-
amylases have been widely used in the baking industry. These enzymes can be added to the
dough of bread to degrade the starch in the flour into smaller dextrins, which are subsequently
fermented by the yeast. The addition of α-amylase to the dough results in enhancing the rate of
fermentation and the reduction of the viscosity of dough, resulting in improvements in the
volume and texture of the product. Moreover, it generates additional sugar in the dough, which
30
improves the taste, crust colour and toasting qualities of the bread. Besides generating
fermentable compounds, α-amylases also have an anti-staling eff.t in bread baking, and they
improve the softness retention of baked goods, increasing the shelf life of these products
Currently, a thermostable maltogenic amylase of Bacillus stearothermophilus is used
commercially in the bakery industry Amylases are also used for the clarification of beer or fruit
juices, or for the pretreatment of animal feed to improve the digestibility of fiber

Textile industry
Amylases are used in textile industry for desizing process. Sizing agents like starch are applied
to yarn before fabric production to ensure a fast and s.ure weaving process. Starch is a very
attractive size, b.ause it is cheap, easily available in most regions of the world, and it can be
removed quite easily. Starch is later removed from the woven fabric in a wet-process in the
textile finishing industry. Desizing involves the removal of starch from the fabric which serves
as the strengthening agent to prevent breaking of the warp thread during the weaving process.
The α-amylases remove sel.tively the size and do not attack the fibres. Amylase
from Bacillus stain was employed in textile industries for quite a long time.

Paper industry
The use of α-amylases in the pulp and paper industry is for the modification of starch of coated
paper, i.e. for the production of low-viscosity, high mol.ular weight starch The coating
treatment serves to make the surface of paper sufficiently smooth and strong, to improve the
writing quality of the paper. In this application, the viscosity of the natural starch is too high
for paper sizing and this can be altered by partially degrading the polymer with α-amylases in a
batch or continuous processes. Starch is a good sizing agent for the finishing of paper,
improving the quality and erasebility, besides being a good coating for the paper. The size
enhances the stiffness and strength in paper Examples of amylases obtained from
microorganisms used in paper industry includes Amizyme® (PMP Fermentation Products,
Peoria, USA), Termamyl®, Fungamyl, BAN® (Novozymes, Denmark) and α-amylase
(Enzyme Biosystems, USA)

31
CHAPTER-3

32
 MATERIAL AND METHDOS
3.1 Isolation and identification of Bacillus sp.: A total of 60 soil samples (from Paonta sahib
sirmour district. A 1g of each soil samples were dissolved in 10 ml sterile distilled water, and
mixed thoroughly. The supernatant of these suspensions was used for isolation of Bacillus
sp.ies which can produce amylase by plating on starch agar at 370C for 24h for amylase
producing bacteria. Enzyme production was identified by clear zone around colonies of
amylase (after addition of iodine) producing bacteria. Isolated predominant, morphologically
distinct colonies were sel.ted and all isolates were identified on the basis of cultural,
morphological and biochemical characteristics.
3.2 Screening of amylase producing organisms: This was carried out on starch agar. This
was then sterilized by autoclaving at 121°c for 15 minutes in an autoclave and then allowed to
cool. The glass Petri-dishes were placed in a canister and sterilized in a hot air oven at 160°C
for 2 hours, the work bench was swabbed with 75% alcohol. After cooling, the efficient
bacterial isolates were re-subcultured onto starch agar plates for det.tion of their enzyme
production efficacies. Productions of these enzymes were studies at various pH and 10) and
temperature A clear zone of hydrolysis on starch (after addition of iodine) gave an indication
amylase producing bacteria. The efficiencies of enzyme production were studied on the basis
of diameter of zone of hydrolysis.
3.3 Extraction of Amylase from the Fermentation Medium: After incubation the
fermentation medium was harvested by centrifugation at 5000 rpm for 20 minutes at 4°C. The
supernatant was coll.ted and subj.ted to estimate the amylase activity. Eff.t of Temperature:
Eff.t of temperature on amylase production the submerged fermentation was carried out at
different temperatures.
3.4 Eff.t of pH: The fermentation medium was prepared by varying the pH values for the
production of amylase, pH in the range of 7.0–10.0 were examined for their eff.t on amylase
production by the sel.ted isolate grown in production media. The pH of the medium was
adjusted using 1 N HCl or 1 N NaOH. The flasks were incubated at 37ºC for 24 h. Samples
were taken at regular time intervals for protein estimation and amylase activity.
3.5 Enzyme production: The inoculum was prepared by inoculating the loopful of strain in to
nutrient broth and it was Incubated in shaker for 24 hrs. 1.5ml of this 24 hr old inoculum was
transferred aseptically to 250 ml production medium 6.0 g Bacteriological peptone; 0.5 g
MgSO4 .7H O; 0.5 g KCl; 1.0 g Starch and incubated in incubator shaker at 180 rpm for 14
33
hrs. The Amylase production was carried out in shake flask fermentation using production
media.
3.6 Enzyme assay Singh et al www.apjhs.com 96 Plate assay: The plate assay was performed
using agar plates amended with starch. The agar plates were prepared amended 2% of starch
with 1.5% of agar. After agar solidification, around 10 mm diameter of well was cut out
aseptically with the help of cork borer. The well was filled with the culture filtrate and
incubated at 37°C for overnight. 1% of iodine solution was over layered on the agar and the
observation was made to see the hydrolytic zone around the well (shown in Figure). The
negative control was maintained by adding sterile water in the separate well.
3.7 Chemical assay: In this method three flasks of 250ml capacity were taken and labeled with
1(starch control), 2(enzyme control), 3(test).After that 5 ml of phosphate buffer was transferred
to each of the flask. Then add 2ml of 0.1% starch solution in the flask 2 and 3, while 2ml of
distilled water in flask 1, and then add of the 2ml of 0.01N HCL in each flask and incubate all
the three flasks at 370C for 3 min. After the incubation, 1ml of active enzyme filtrate was
added in the flasks 1 & 3 and 1ml of the inactivated (heat killed) enzyme in flask 2.incubate at
370 C in water bath for 15, 30, & 45 min. Thereafter add 80 ml of distilled water in each flask.
Then 4 ml of 0.01N iodine in each flask was added, mix well and measure absorbance at
578nm.And the enzyme activity was calculated by the formula Volume activity 12.35
(constant) V= Volume of starch The amylase activity was determined in IU/mL/min by
applying the following formula (6). Activity of enzyme × 1000 IU/ml/min = Mol.ular wt. of
maltose × time of incubation
3.8 Purification of Amylases: After 24 hrs of growth, the culture was centrifuged at 5,000
rpm for 5 min at 4°C and the cell free supernatant was used .Amylase produced was partially
purified by pr.ipitation with ammonium sulphate and followed by dialysis. Ammonium
sulphate pr.ipitation t.hnique was performed by mixing culture filtrate and ammonium sulphate
(75%, w/v) solution at 1:3 ratios [7]. The mixture was then stored in cold room for 24 h to
pr.ipitate all the proteins present in the sample. Pr.ipitate was removed by centrifuging sample
in an ultra centrifuge at 10000 rpm for 10 min. The supernatant was discarded and pr.ipitate
obtained was dissolved in 5 ml of 1 M-citrate phosphate buffer (pH.5) (8). Then the mixture
was subj.ted to dialysis. The enzyme was dialysed in same buffer overnight at 4°C.

34
CHAPTER-4

35
 RESULTS

RESULTS: Total 60 samples were coll.ted from 20 different places of Paonta sahib. Out of 60 samples
17 isolates screened tested positive. The isolates were characterized and identified on the basis of
Morphological characteristics such as Gram staining, Colony characteristics and Biochemical test Holt
et al.1994.Bergey’s manual of determinative Bacteriology, 9th Ed. Based on the Morphological &
Biochemical characterization. In present study On the basis of level of productivity of the amylase, an
isolate producing a maximum of amylase activity was screened from soil and used for detailed
investigation. Screening of the amylase producing organisms was carried out on the starch agar.
Productions of these enzymes were studies at various pH and temperature. A clear zone of hydrolysis
on starch (after addition of iodine) gave an indication amylase producing bacteria the efficiencies were
studied on the basis of the zone of hydrolysis. The isolate showing the maximum zone of hydrolysis
was sel.ted for identification. The use of starch nutrient agar and iodine for det.ting amylase (hydrolytic
enzyme) producing Microorganisms have been reported by Forgarty and Kelly, (2018) and also by
Iverson and Millis, (2019) that starch hydrolysis can be det.ted on plates as a clear zone surrounding a
colony. This procedure employed showed a positive result for the Aspergillus strain isolated. The
m.hanism of clear zone observed was due to the fact that the amylase produced during the growth of the
organisms has hydrolysed the starch around the colony, thereby testing negative when flooded with
iodine. The un-hydrolysed part of the plate tested positive to the presence of starch (amylose), hence the
blue-black appearance. According to Akpan et al. (2020) screening for amylase producing
microorganism by the www.apjhs.com 2019 method described above is time consuming and
inconvenient for dir.t isolation of intact cells, as the cells die after flooding with iodine, therefore a
rapid screening method such as Remazol Brilliant Blue (R.B.B) will be more eff.tive. The former
method was adopted for this research which involves screening through the use of starch agar and
iodine solution. The formation of clear zones indicated that the organisms test positive for 17 isolates
screened. Table 1: Biochemical Characterization Eff.t of incubation temperature The data of tables
shows the eff.t of different incubation temperatures on the production of amylase by Bacillus sp. In the
present study 17 isolates of Bacillus sp.ies produced enzymes over the large range of temperature
however the maximum zone of starch hydrolysis observed at temperature 400C. Below 350 C showed
d.rease in the zone of starch hydrolysis. Fig. 5.4 shows the activity of enzyme was gradually increased
and found maximum at temperature zone of starch hydrolysis of isolate I-8. On the basis of the
maximum zone of starch hydrolysis I-8 was sel.ted for the quantitative analysis of enzyme. The
fermentation was carried out at different temperatures

36
CHAPTER-5

37
 DISCUSSION

In present study On the basis of level of productivity of the amylase, an isolate producing a
maximum of amylase activity was screened from soil and used for detailed investigation.
Screening of the amylase producing organisms was carried out on the starch agar. Productions
of these enzymes were studies at various pH and temperature zone of hydrolysis on starch
(after addition of iodine) gave an indication amylase producing bacteria the efficiencies were
studied on the basis of the zone of hydrolysis. The isolate showing the maximum zon
hydrolysis was sel.ted for identification. The strain was Gram +ve, rod shaped aerobic,
Catalase +ve, MR +ve, CU +ve and VP +ve. The bacterial isolate was identified as Bacillus
subtilis. Cordeiro et al., 2021 showed sp.ies produce a large variety of extra cellular enzymes,
such as amylases, which have significant industrial importance. The use of starch nutrient
agar and iodine for det.ting amylase (hydrolytic enzyme) producing microorganisms have
been reported by Forgarty and Kelly, (2019) and also by Iverson and Millis, (2017) that starch
hydrolysis can be det.ted on plates as a clear zone surrounding a colony. In present study
production of amylase by bacillus subtilis (36 IU/mL) with pH 7 at 40°C for 14 hrs. by Sekar
Sudharhsan, et al., Amylase producing sp. was isolated from spoiled food waste, which
yielded 30 U ml-1 of amylase in medium containing 4% starch and 2% yeast extract at 37°C,
pH 7.0 after 20 h of incubation. Maximum amylase activity was at pH 7.0 and 37°C. The
enzyme retained 70% activity at pH 9.0 Riaz, et al., 2016 reported the maximum production
of enzyme was optimized at the pH 7.5, while the incubation temperature investigated was
40ºC,

38
CHAPTER-6

39
 CONCLUSION

The use of α-amylase in starch based industries has been prevalent for many d.ades and a
number of microbial sources exist for the efficient production of this enzyme, but only a few
sel.ted strains of fungi and bacteria meet the criteria for commercial production. The search
for new microorganisms that can be used for amylase production is a continuous process.
More r.ently, many authors have presented good results in developing α-amylase purification
t.hniques, which enable applications in pharmaceutical and clinical s.tors which require high
purity amylases.

40
CHAPTER-7

41
 REFERENCES

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