DA NANG UNIVERSITY OF SCIENCE AND TECHNOLOGY

FACULTY OF CHEMICAL ENGINEERING – BIOTECHNOLOGY DIVISION

Cellulase
Cellulose Degrader

Instructor: PhD. Nguyen Hoang Minh

1
 MEET OUR TEAM

Hung Qua Thu

Dat Nam Khang 2

 Table of Contents

Specific
Overview Conclusion References
Application
Definition, Sum up all the All the things we
classification, An article of a
importances need to carry out
structure, sources potential application
this spectacular
of celluase
slide
 Overview
Definition, classification,
structure, sources

1
OVERVIEW

CELLULASE

o Cellulases are enzymes that catalyze the hydrolysis of

the 1, 4 linkages present in the linear glucose
polymer, cellulose (EC #3.2.1.4). They are members of a
class of enzymes called glucoside hydrolases and these
enzymes are cataloged into sequence related families on
the CAZy.
o Cellulase has a wide range of applications in Industrial
Biotechnology and is the second most used industrial
enzyme after protease. In most of the cases, they are used
in combination with other enzymes like pectinase,
hemicellulase, ligninase,…

5
OVERVIEW

SOURCES FOR PRODUCTION FUNGI

o Most of the cellulases exploited for industrial

applications are from filamentous fungi such as
Trichoderma, Penicillium, Fusarium, Humicola etc.
o The most emphasis has been placed on the use of fungi
because of their capability to produce copious amounts
of cellulases and hemi cellulases which are secreted to
the medium for easy extraction and purification.
o In addition, fungal enzymes are often less complex
than bacterial glycoside hydrolases and can therefore
be more readily cloned and produced via
recombination in a rapidly growing bacterial hosts such
as E. coli.

6
OVERVIEW

SOURCES FOR PRODUCTION BACTERIA

o Cellulases of bacterial origin are less studied because

fungi are known to be better producers of these
enzymes.
o Both aerobic and anaerobic bacteria are known to
produce cellulase. Profound differences are noted
between aerobic and anaerobic microorganisms
regarding their cellulase system, cell mass yield and end
products of biomass degradation.
o Most of the bacterial cellulolytic enzymes are reported
from Bacillus, Acinetobacter, Cellulomonas and
Clostridium.

7
OVERVIEW

SOURCES FOR PRODUCTION EXTREMOPHILES

o Extremophiles offer a unique environment that allows the use of a biocatalyst obtained from these
sources for application in specific fields where the physical factors such as pH, temperature, ionic
strength, acidity and alkalinity are at their extremes.
o Thermophilic cellulose-degrading bacteria have been isolated from various environments such as
compost systems, soils, wastewaters, and from the deep subsurface of gold mines.
o A large number of bacterial and archaea species across different genus such as Bacillus, Clostridia,
Fervidobacterium, Rhodothermus, Thermoplasma, Thermotoga, Pyrococcus, Sulfolobus,
Thermococcus, Desulfurococcus have been reported to produce thermostable cellulases with diverse
properties.

8
 Structure

Cellulase is a complex system of enzymes, including:

Endo-1,4-β-D-glucanase = Endoglucanase = Carboxymethyl cellulase (CMCase)

Exo-1,4-β-glucanase = Exoglucanase = Exocellobiohydrolase = Cellobiohydrolase (CBH)

β-D-glucosidase = β-D-glucoside glucanohydrolase = β-Glucosidase (BG) 9

Cellulose hydrolysis mechanisms

Endoglucanases hydrolyze bonds of cellulose

chains randomly to produce new chain ends.

Exoglucanases cleave cellulose chains at the

ends to release soluble cellobiose, cellodextrin or
glucose

β-glucosidases hydrolyze cellobioses and

cellodextrins to glucose in order to eliminate
cellobiose inhibition

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Phases of Hydrolysis
Primary hydrolysis:
- Occurs on the surface of solid substrates

- Performed by endoglucanases and

exoglucanases

- Is the rate-limiting step for the whole cellulose

hydrolysis process

Secondary hydrolysis:
- Occurs in the liquid phase

- Involves primarily β-glucosidases

- Although some β-glucosidases also hydrolyze

longer cellodextrins

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Production
technologies

1
Production technologies

SSF

Solid state fermentation (SSF) is defined as the

fermentation in the absence or near absence of
free water, although the substrate retains
enough moisture to allow growth and support
metabolism of the microorganism

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 Production technologies

SmF

Submerged fermentation (SmF) has been defined as

fermentation in the presence of excess water. SmF is a simplistic
process and provides better control

The filamentous fungi T. reesei or A. niger are frequently used in

commercial SmF cellulase production

Production of cellulase through SmF can be influenced by the

nature of the cellulosic substrate, pH, the medium’s temperature,
and nutrient availability

14
Research at
home and abroad

1
 Domestic Research

Currently, research on cellulase enzyme in water is having the

following changes:
Application of cellulase enzyme in production: Research shows
that cellulase enzyme can be effectively used to produce
biological products such as functional foods, animal feed,
biological materials,...
Searching for new cellulase enzymes: Researchers are looking
for cellulase enzymes that are more efficient and work at
different temperatures and pH levels for wider application in
many fields

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 Research Abroad

Research on cellulase enzymes abroad often focuses on

issues related to the application of cellulase in papermaking,
sugar production, beer production and biofuel production.
Research also focuses on optimizing enzyme production
conditions, increasing enzyme stability and durability, as well
as improving production efficiency and reducing production
costs.
In addition, the research focuses on exploiting new species
of bacteria and fungi to produce cellulase enzymes that are
able to work better under extreme conditions.

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 Research Abroad

• "Cellulase immobilization on graphene oxide: Kinetic, thermodynamic, and thermomechanical

analyses." (2021)
• "In situ cellulose dissolution by cellulase-mimetic metal-organic frameworks." (2020)
• "Strategies for improving cellulase production in microbial hosts: Current state and prospects."
(2020)
• "Expression and characterization of a novel cellulase gene (cel8B) from the gut microbiota of the
termite Reticulitermes chinensis." (2018)
• "Fungal Cellulases and Their Applications in Industries" (2020)
• "Cellulases from Marine Microbes and Their Applications in Biotechnology" (2020)
• "High-yield production of cellulase from Aspergillus niger by solid state fermentation and its
application in saccharification of waste newspaper" (2021)
• "Purification and characterization of cellulase produced by Aspergillus niger KIBGE-IB36 and its
application for the hydrolysis of agro-wastes for bioethanol production" (2021) 18
Measurement
Methods
 Measurement methods
Quantitative assays

• Reducing sugars: DNS, BCA, PAHBAH,..

Hydrolysis products • Total soluble sugars: phenol - H2 SO4
• Chromophores.

Reduction substrate • Gravimetry methods

quantity • Chemical methods.

physical properties of • Viscosimetric determinations

substrates • Turbidity measures
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 Cellulase activity assays
-Based on a reduction in
substrate viscosity and/ or
an increase in reducing
ends.
-On agar plates (CMC,
cellulose) with various dyes.
-p-nitrophenyl glucoside
and methylumbelliferyl-β-D–
glucoside.
Endogulacanase
Measurement methods
-Avicel has been used for -Based on colored or -Using insoluble substrates
measuring exoglucanase fluorescent products -Pure cellulosic substrates
activity. released from pnitrophenyl (Whatman No. 1 filter paper).
-4-methylumbelliferyl-β-D- β-D-1,4-glucopyranoside -Microcrystalline cellulose.
lactosideis an effective -can be measured using -The most common is the
substrate. cellobiose and longer filter paper assay (FPA) using
cellodextrins. Whatman No. 1 filter paper
Exogulacanase 𝜷 - D - glucosidases Total Cellulase
22
 Specific
Application
An article of a potential
application of celluase
Application of Cellulase from an
Alkalothermophilic
Thermonospora sp. in
Biopolising of Denims
Rationale
Concern, solution
 Concern

Acid cellulase Neutral cellulase

(Trichoderma ressei sp.) (Humicola isolens sp.)

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Concern

Practical experiments indicated that pH

of 6 and beyond would prevent
back-staining efficiently.
=> Seek for an enzyme which is active
at alkaline pH

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 Solution

• Higher abrasive activity

Alkaline Cellulase
• Lower backstaining possibility
(Thermomonospora
• Effectiveness under non-buffering conditions
sp.) (T – EG)
=> Meaningful application in textile industry

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Treating process
Activity assay, optimal
conditions
Mechanism

a) Bare fabric = protruding fibers

+ cellulose
b) Dye thecolor -> protruding
fibers are expose
c) Usingenzyme -> cut the fibers
+ cellulose
d) Got the desired fabric that you
want
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 Enzyme Activity Assay

Determination

CMC
Enzyme Buffer
subtrate

• Reducing sugar ->

dinitrosalicylicacid
• Protein -> Bradford

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 Identification of Optimum pH,
Temperature, and Stability of endoglucanase

pH stability Enzyme

Temperature
Enzyme
stability

31
 Treating Fabric

-Trichoderma reesei
Fabric is cellulase: pH =5
Under -Humicolacellulase:
treatedwith buffering pH = 7
different -T-EG: pH =8
concentrations
of T-EG and
Trichoderma + Under
Humicola -T-EG : normal water
non- (pH = 7.8)
cellulase buffering

32
Assessment
Methods
Hariness Measurement,
Cellulase Abrasive
Activity...
 Hariness Measurement

Determination of
the number of Obtaining average
hairs and hair statistics
length

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 Measurement of Abrasive and Backstaining
Activity

Calculating
Abrasive average
activity value of
color

Analyzed and
Backstaining evaluated for
activity color
intensity

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 Measurement of Softness and Subjective
Assessment of Performance of Enzyme

Calculating bending
Softness length and flexural
rigidity

reducing hairiness,
Subjective Assessment impartation of
of Performance of softness, wash-down
Enzyme effect, backstaining,
color contrast

36
Results
& Discussion
Under buffering and also
non-buffering
 Enzyme Activity
Assay
• The maximum endoglucanase
production of 12 IU/ml.
• Had pH stability in a broad
range (4-10). It was most
stable at pH 8.
• It was thermostable showing
a half life of 20 min at 80 C at
a protein concentratuon of 50
mg.mL

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 Denim Biofinishing Under Buffering Conditions

Hairiness of the
Fabric

A steady decrease in hairiness of the fabric is seen on increasing the dosage of the
enzyme up to 1.5%. It is also observed to be as effective as Humicola cellulase and
better than Trichoderma cellulase.
39
Abrasive activity
& Backstaining

T-EG exhibits higher abrasive

activity as compared to Humicola
cellulase with a marginally lower
backstaining index.

40
Softness of the Fabric

The flexural rigidity of the fabric is inversely

proportional to the softness of the fabric

41
 Denim Biofinishing Under Non-Buffering Conditions

Abrasive activity
& Backstaining
T-EG shows better abrasive
activity than the commercially
used enzyme, Humicalo
cellulase. There as a marginally
lower backstaining activity is
exhibited by T-EG as compared
to Humicola cellulase.

42
Sum-up

● Use of Thermomonospora sp. cellulase rich in endoglucanase and having

negligible activity towards crystalline cellulose.
● Activeness and stability under alkaline condition appears to be an excellent
alternative for biostoning and biofinishing of denim and compare well with
commercial cellulases under alkaline conditions.
● T-EG caused less backstaining and is effective under non-buffering condition
which is more preferred for industrial application.

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 Conclusion
• Cellulase plays a crucial role in the degradation of cellulose, which is the most
abundant organic compound on earth.
• It is an enzyme system consisting of multiple types of enzymes working
together to break down cellulose into simpler sugars that can be utilized by
organisms.
• Cellulase has numerous industrial applications, including biofuel, paper
manufacturing, food and beverage, and textile processing.
• The study of cellulase and its mechanisms of action is ongoing, new
applications and uses for this enzyme are continually being discovered.
 References
• Anoop Kumar, V., Suresh Chandra Kurup, R., Snishamol, C., & Nagendra
Prabhu, G. (2019). Role of Cellulases in Food, Feed, and Beverage Industries. In
Energy, Environment, and Sustainability (pp. 323–343). Springer NatureIt
• R. Anish, M. S. Rahman, and M. Rao, “Application of cellulases from an
alkalothermophilicThermomonospora sp. in biopolishing of denims,”
Biotechnol. Bioeng., vol. 96, no. 1, pp. 48–56, Jan. 2007.
• S. P. George, A. Ahmad, and M. B. Rao, “Studies on carboxymethyl cellulase
produced by an alkalothermophilic actinomycete,” Bioresource Technology, vol.
77, no. 2, pp. 171–175, Apr. 2001
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