RESEARCH PAPER

CELLULASE ENZYME

Synopsis by
Diganshi Verma
Acharya Narendra Dev Nagar Nigam Mahila Mahavidyalaya, Kanpur

A Research Paper
Submitted in Partial Fulfillment of the Requirements for the
Master of Science Degree in the
Field of Chemistry

Supervised by:
Professor Arti Saxena
H. O. D of chemistry department
Acharya Narendra Dev Nagar Nigam Mahila Mahavidyalaya, Kanpur
 Acknowledgment

I wish to express my gratitude towards Prof. Arti Saxena, Head of department of chemistry
A.N.D.N.N.M.Mahavidyalaya, Kanpur for her sincere co-operation and supervision throughout
my project. I am also grateful to the other teachers of the chemistry department Dr. Sarika
Yadav, Mrs Shikha Mishra and Miss Jyoti Singh for their Kind support. I am highly obliged to
the non teaching staff of the Department for helping at various stages throughout the execution of
this project work. At last but not the least thanks to my classmates for their affectionate co-
operation.

Diganshi Verma
 Table of content

• Abstract
• Introduction
• Cellulase degradation
• Mechanism of cellulose hydrolysis
• Cellulose recovery and purification
• Optimization of physiological factors
• Application of cellulase
• Conclusion
• Future outlook
• References
 ABSTRACT

Cellulase enzymes play a crucial role in the efficient degradation of cellulose, a major
component of plant biomass. These enzymes, produced by a variety of microorganisms, are
capable of hydrolyzing the β-1,4-glycosidic bonds in cellulose, liberating glucose and other
fermentable sugars. This abstract provides an overview of the structure, function, and
biotechnological applications of Cellulase enzymes.
Cellulases are categorized into three major classes: endoglucanases, exoglucanases, and β-
glucosidases. Endoglucanases randomly cleave internal β-1,4-glycosidic bonds within cellulose
chains, while exoglycanases act on the cellulose ends, liberating cellobiose or glucose. Β-
glucosidases complete the process by hydrolyzing cellobiose to glucose. The synergistic action
of these enzymes is crucial for efficient cellulose degradation.
The structure of cellulase enzymes varies widely, with catalytic domains exhibiting different
folds and arrangements. Cellulases often possess carbohydrate-binding modules (CBMs) that aid
in substrate recognition and binding. Elucidating the structure-function relationships of these
enzymes has enabled researchers to engineer and optimize cellulases for enhanced activity and
stability.
Cellulase enzymes have found numerous applications in various industries. They are extensively
utilized in biofuel production, where they assist in the hydrolysis of cellulose to fermentable
sugars, which can be subsequently converted into bioethanol or other valuable products.
Additionally, cellulases are employed in the textile, paper, and food industries to enhance fiber
processing, improve paper pulp quality, and facilitate the extraction of juices from fruits,
respectively.
The biotechnological potential of cellulase enzymes is further enhanced by advancements in
genetic engineering and enzyme immobilization techniques. Engineered cellulases with
improved thermal stability, pH tolerance, and substrate specificity have been developed.
Immobilization methods such as enzyme encapsulation and immobilization on solid supports
allow for their reuse, reducing production costs and improving process efficiency.
In conclusion, cellulase enzymes are key players in cellulose degradation, offering immense
biotechnological potential. Understanding their structure, function, and applications has paved
the way for their optimization and utilization in various industries. Continued research in this
field holds promise for the development of sustainable and efficient bioconversion processes and
the advancement of a bio-based economy.

INTRODUCTION
Cellulose
Cellulose is a main renewable form of carbohydrates, with about 1011 tons synthesized Annually.
It is an unbranched β-1, 4-linked homopolymer of glucose, but cellulose have lots Of variation in
their chain lengths according to origin and in the degrees of interaction Between chains.
Cellulose is an important component of the primary cell wall of Angiosperms and algae. Some
species of bacteria secrete it into biofilms. Cellulose is the most Abundant organic polymer on
earth. The cellulose content of cotton fiber is 90%, wood is 40–50% and dried hemp is about
45%. It has the greatest potential for bioethanol production. However, cellulose must be
hydrolysed to obtain fermentable sugars and the cellulolytic Enzymes are central to the
processing of biomass for bioethanol production. [1]
The Source of the cellulose in plants is found as microfibrils (2-20 nm in diameter and 100-40,
000 nm long). Annually, plants produce 4×109 Tons Cellulase. It is one of the most important
source of carbon on earth and its biosynthesis occurs at a rate of 0.85×1011 tons per annum by
both land plants and marine.[2]

Cellulase
Cellulases are a group of enzymes produced primarily by fungi, bacteria, and protozoans that
Hydrolyse cellulose. Termites and microbial intestinal symbionts of some termites also Produce
cellulase. Several kinds of cellulases are structurally and mechanistically different.[3]
Cellulase is an important class of enzyme that catalyzes the hydrolysis of a single substrate. It
degrades insoluble cellulose to soluble Sugars. Cellulase is a polysaccharide linked with β-1, 4-
glucosidic bond. It is composed of three enzymes: 1, 4 – β-glucanase and β-glucosidase.
Exoglucanase cleaves at the non reducing end of cellulose chain; splits the elementary fibres
from crystalline Cellulose and β-1, 4-glucosidase hydrolyse cellobiose and cellodextrins (water
soluble) to glucose. Cellulase plays an important role in producing fermentable sugar from
Lignocellulosic biomass. It is low-cost, sustainable feedstock for the production of biochemical
as it is rich in cellulose content. It comprises of Hemicellulose (20-30%), cellulose (30 – 40%),
and lignin (20-30%). The hydrolysis of lignocellulosic material to monosaccharides is a major
problem. Cellulase is Extracted from the gut of microorganism like termites (Isopteran) and
bookworm (Lepidoptera) thriving on cellulosic biomass as major feed. The Microbial species
(termites) plays an important role in the recycling of photosynthetically fixed carbon. Fungi
(anaerobic cellulolytic) and bacteria (may Be aerobic, anaerobic, thermophilic and mesophilic)
are the major source in the production of cellulases. A few species of genera Aspergillus,
Penicillium and Trichoderma are used to obtain lignocellulosic biomass.[4]

Types and mode of action

Cellulase is composed of five type of enzyme system based on their mode of action.
• Endocellulase – It randomly cleaves internal bonds at amorphous sites and creates new
Chain ends.
 • Exocellulase – It cleaves two to four units from the ends of the exposed chains produced
By endocellulase, resulting in formation of cellobiose.
• Cellobiase or beta-glucosidase – It breaks down the product of exocellulase enzyme into
Individual monosaccharides.
• Oxidative Cellulases – It depolymerizes cellulose by radical reactions, as for instance
Cellobiose dehydrogenase.
• Cellulose Phosphorylases – It depolymerize cellulose using phosphates instead of water.

Figure 1. Diagrammatic overview represents the cellulose hydrolysis by cellulase system.

Cellulose structure and properties

Cellulolytic enzymes are the third most important industrial enzyme due to its versatile
applications in Various industries such as paper and pulp, textile and detergent industry. The
resurgence in utilization Of biomass for bio-ethanol and other value added organic compounds
production has attracted major Attention of researchers globally towards cellulases.
A large group of naturally occuring microorganisms, fungi and bacteria, have the ability to
degrade Lignocellulosic materials to its building components. More than 100 cellulolytic fungi
have been Reported to date and this number is continually increasing, including (hyphomycetes,
ascomycetes, And basidiomycetes), as well as many filamentous bacteria species. Cellulolytic
microorganisms are able to digest a variety of other carbohydrates besides cellulose. Another
property of these organisms is the ability of secreting large quantities of extracellular protein Into
the medium[5]

CELLULOSE DEGRADATION
• Cellulose degrading enzyme
Cellulolytic enzymes play an important role in natural biodegradation processes in which plant
Lignocellulosic materials are efficiently degraded by cellulolytic fungi, bacteria, actinomycetes
And protozoa. In industry, these enzymes have found novel applications in the production of
Fermentable sugars and ethanol, organic acids, detergents and other chemicals. Cellulases
Provide a key opportunity for achieving tremendous benefits of biomass utilization. The
conversion of cellulose into glucose is now known to consist of two steps in the enzyme System
of Trichodermaviride. In the first step, beta-1,4glucanase breaks the glucosidic linkage To
cellobiose, which is a glucose dimer with a beta-1, 4 bond as opposed to maltose, a Counterpart
with an alpha-1, 4 bond. Subsequently, this beta-1,4glucosidic linkage is broken by Beta-
glycosidase:
b-1,4 glucanase b-glucosidase
Cellulose -------------→Cellobiose -----------→ Glucose

• Cellulose degrading microorganisms

Cellulolytic enzymes are synthesized by a number of microorganisms. Fungi and bacteria are the
Main natural agents of cellulose degradation. The cellulose utilizing Population includes aerobic
and anaerobic mesophilic bacteria, filamentous fungi, thermophilic And alkaliphilic bacteria,
actinomycetes and certain protozoa. However, fungi Are well known agents of decomposition of
organic matter, in general, and of cellulosic substrate in particular.
Microorganisms bring about most of the cellulose degradation occurring in nature. Aerobic
bacteria Produced numerous individuals and extra-cellular enzymes with binding modules for
different Cellulose conformations, while anaerobic bacteria possess a unique extracellular multi
enzyme Complex, called cellulase. However, the main cellulose utilizing species are the aerobic
and Anaerobic hemophilic bacteria, filamentous fungi, basidiomycetes, thermophilic bacteria and
Actinomycetes. At the first step, the microorganisms responsible for cellulose Decomposition
bring about an enzymatic hydrolysis of the complex polymer, that is, the enzymes System which
involves a group of different enzymes, is collectively known as cellulase.
Mainly efficient cellulase activities are observed in fungi but there is increasing interest in and
cellulase production by bacteria because bacteria have high growth rate as compared to fungi and
has good potential to be used in cellulase production. The search for a novel and improved
Bacterial strain, having hyper cellulase productivity with more activity and high stability against
Temperature, pH and under non-aseptic conditions might make the process more economical.[6]

MECHANISM OF CELLULOSE HYDROLYSIS

Cellulolytic microorganisms play an important role in the hydrolysis of lignocellulosic polymer

materials. Cellulase enzyme breaks down cellulose into Monosaccharides using three major
enzymes: Endoglucanase, exoglucanase and β-glucosidase. Endoglucanase randomly cleaves at
internal site of Cellulose to yield oligosaccharides of different lengths while on the other hand,
exoglucanase attacks on the reducing and non-reducing ends of cellulose to Liberate glucose,
cellobiose and cellooligosaccharides hydrolyzed by β-glucosidases.[7] The efficient hydrolysis
requires presence of β-glucosidases as it Is inactive against amorphous and crystalline cellulose.
The end products of endoglucanase and cellobiohydrolases are cellodextrans and cellobiose
Respectively which inhibit the enzyme activity.[8] The mechanism of hydrolysis is shown in
figure.

The three types of reactions catalyzed by cellulases:

(1) Breakage of the non covalent Interactions present in the amorphous structure of cellulose by
endoglucanase
(2) Hydrolysis of Chain ends to break the polymer into smaller sugars by exoglucanase
(3) Hydrolysis of Disaccharides and tetrasaccharides into glucose by β-glucosidase

CELLULASE RECOVERY AND PURIFICATION

After the production of the cellulolytic enzymatic complex, it has to be separated and purified in
order To obtain a stable product. The separation process in this case involves removal of
cellulolytic Enzymes from the medium components, polysaccharides, small molecules and
proteins other that Cellulolytic ones.
The purification process involves four main stages:
• Removal of insoluble components (solids),
• Recuperation of the protein and purification in order to increase their concentration
 • Separation of different fractions of the extracellular enzymes (usually chromatographical
Methods)
• Preparation of the pure enzyme, in a final form that is ready for commercial use

Separation and purification processes vary greatly when it comes to performance, price, and
Applicable scale. As defined by Stanbury et al. [Stanbury et. Al, 1984.], there are several criteria
when It comes to determining the optimal recovery process:
1. Location of the product: intra- or extracellular
2. Concentration of the product in the fermentation broth
3. Physical and chemical properties of the product
4. Intended use of the product
5. Minimal acceptable purity level
6. Magnitude of bio-hazardous elements in the broth
7. Impurities in the fermentor broth
8. Product’s marketable price
Some most commonly used enzyme separation and purification techniques are listed, Along with
the exact property of the enzyme that is the basis on which the separation is made.

OPTIMIZATION OF PHYSIOLOGICAL FACTORS

Cellulase activity is enhanced by various physiological factors like pH, temperature, substrate,
carbon source, and nitrogen source and incubation period. These should be optimized over a
wide range. [9]

A. Effect of pH and temperature:

The pH and temperature play a key role in the enzyme production. Their optimization has to be
done as it affects the production of enzyme directly. The Cellulase activity has been recorded at
pH value 5.0 at temperature 30°C for Penicillium and pH value 4 at temperature 30°C for
Aspergillus tubingensisKY615746 .

B. Effect of carbon source:

The carbon sources such as starch, glucose, maltose, lactose and fructose has been used to
replace glucose (original carbon source). Glucose has been Documented as the highest cellulase
production. It has been found to be significant source for production of cellulase enzyme.
Ishihara et al. studied the Utility of source and found sucrose, glucose and mannitol as suitable
source for optimum levels of cellulase production.

C. Effect of Nitrogen:
The nitrogen source are yeast extract, peptone, urea and ammonium sulphate, ammonium
chloride. The extracellular cellulase production has shown Sensitivity towards nitrogen sources.
Mandrels et al. observed the effect of nitrogen in the growth medium. Among nitrogen sources
ammonium Sulphate has been recorded as good nitrogen source. The maximum activity of
cellulase has been obtained on yeast extract and combination of peptone And ammonium
molybdate.

D. Effect of incubation period:

The time taken by microorganism to synthesize enzyme by using medium nutrients is termed as
incubation period. It depends on the type of fermentation Technique and microorganism used. As
SSF take longer time in comparison with SmF. Therefore, bacteria requires less time than fungi
for cultivation. Ahmed et al. has been reported 192 hrs and 168 h suitable for fermentation using
Myceliophthora heterothalika containing wheat bran and sugarcane.

APPLICATIONS OF CELLULASE
Cellulase is widely used in various industries such as food, animal feed, agriculture and biofuel,
textile and laundary. These are discussed below-
• Food processing industry

Cellulase plays an important role in food industry. It is used to improve nutritive quality of
fermented food items and in production of low calorie food Ingredient oligosaccharides. It is
utilized in the production of food colouring agent as carotenoid which is a group of colouring
substance responsible for Many plant colours from red to yellow. Macerating enzyme consists of
cellulases with pectinase and hemicellulase are used to improve the cloud Stability and texture of
nectar and purees. Cellulase and pectinase are used to disrupt the cell wall of potato, carrot and
orange peel and release Carotenoid. It is also used to release antioxidants from fruit and
vegetable pomace and helps in controlling atherosclerosis, coronary heart disease and Reduce
food spoilage.

• Animal feed industry

The animal feed industry is an important sector of agribusiness comprising poultry, pigs,
ruminants, pet foods and fish farming. Cellulase is utilized to Improve feed, body weight and
feed gain by ruminants. Cellulase degrades certain cereal component and upgrades the nutritional
value of feed. The Dietary fibres present in feed consist of non – starch polysaccharides such as
arabinoxylans, cellulose, other plant component including chitin, β-glucan, Waxes, lignin, and
oligosaccharides like proteases, amylases and glucanases. In the feed of monogastric animals, β-
glucans which helps in decreasing Intestinal viscosity and release nutrients from plants (grains),
enhance the digestion and absorption of feed and weight gain by chickens and boilers. Cellulase
in addition with proteases is significant to improve the quality of pork meat.
• Textile and laundry industry
Cellulase is the third largest group of enzymes used in textile and laundry. It is used to improve
the fabric quality by biostoning of denim garments, Biopolishing of non-denim fabrics,
defibrillation and biofinishing. In biostoning process, cellulases act on cotton fabric and break
down small fibre Ends results in loosening the dye after washing. This process includes the fibre
damage, less work intensive. It increases productivity of machine. The fibres such as cotton,
linen, viscose, ramie, and lynocell (pure cellulosic fork of wood) are used in the manufacture of
fabric. These fibres form fuzz as Well as pilling results in negative impact of cellulosic fibres.
Therefore, biopolishing is used to overcome this problem by bleaching, desizing, Scouring,
bleaching and dying. Cellulase is also used in biofinishing as it is rich in endoglucanase activity.
Cellulase along with protease and lipase in Detergents widely used as household washing
powder. Cellulase is applicable to remove colour brightness, softness in cotton fabrics and helps
in Removing soil from inter fibril spaces.
• Brewery industry
Cellulase is used in the production of wine and beverages. In wine production, the enzyme such
as pectinases and glucanases and hemicellulases play a Key role by removing colour extraction
skin maceration, filtration and improve the stability and quality of wine. Macerating enzyme also
improves Settling, press ability and juice yield of grapes for fermentation. Brewing is based on
the action of enzyme activated during malting and fermentation. Malting mainly depends on
germination of seed which initiates the activation of amylase, biosynthesis carboxypeptudase and
cellulases to produce high Quality malt.
• Paper and pulp industry
Cellulase has broader applications in paper and pulp industry. It is one of the largest industrial
sectors. Cellulase is used for bio-mechanical pulping, bio-Modification of fibres, removing of
ink coating and toners from paper, improving drainage of paper mills. It is also used in the
manufacture of soft paper Like sanitary paper, paper towels and biodegradable cardboard.[10]
Cellulases are also used to enhance the bioleachability of soft wood kraft pulp. The cellulase
alone or combination of cellulose and hemicellulase has been used to improve pupil betability,
runnability, paper sheet and trouble free Printing process. Cellulase in addition with xylanase is
considered as most affective for recycling the waste paper from old books, newspapers,
magazines. This process is known as deinking. It leads to the formation of newspaper or ethanol.
The enzymative deinking has ability to improve fibre brightness, Pulp free and cleanliness,
enhance strength properties and reduce fine particles in the pulp. Deinking also prevents the
alkaline yellowness, Change the ink particle size by using cellulases at acidic pH.
• Agriculture industry
Cellulase is used for many purposes in agriculture industry. It is used for increasing crop growth
and in improvement of soil fertility. The use of Exogenous cellulase is a potential means to straw
decomposition and increase soil fertility. Straw incorporation is strategy to improve soil quality
and Reduce the dependence on mineral fertilizers. Cellulase is also used as biocontrol agents to
provide protection against plant pathogens. [11]
• Biofuel industry
Cellulase is very important in the biofuel production. The consumption of fossil fuels and
increasing demand of alternative renewable source of energy has Developed a huge interest in it.
The bioconversion of lignocellulosic substrates and other enzymes are used for the commercial
production of biofuel. Cellulase is used in the conversion of non food biomass such as
agriculture waste and energy crops into fermentable sugar for renewable fuel and Chemical. The
use of agro wastes such as sugarcane, bagasse, rice straw for bioethanol production are
invaluable and do not create any problem in the Environment.

CONCLUSION

Currently, cellulase is gaining huge attention of industries and causing an enormous economic
impact. With a view to develop an economically feasible technology for growing demand of
cellulase and also to realise its potential in biotechnological research is very crucial. Various
industries favour the Technology having low cost, readily available carbon sources (agro wastes)
and eco-friendly strategies. So, cellulase can be modified for the effective Conversion of easily
available agro waste biomass into fermentable sugar, fuels and other products. Therefore,
appropriate methodology has to be Developed for producing high enzyme titre at low cost and
also eco-friendly in nature. Enzyme-based industries are gaining importance over the chemical-
based industries due to process safety, low refining cost, high yield, efficient process control and
friendly nature. Enzymes, particularly cellulases, have potential applications in paper,
pharmaceutical, detergent and food industries. Thermostable cellulases have extensive use
because of their stability at elevated temperature. The most promising application of cellulases
nowadays are in the beverage, feed and food industries. We would like to emphasize again the
importance of research on cellulases and their applications in the food industry. It is particularly
important to cater to the diversified needs of food industry.[12]

FUTURE OUTLOOK

Although cellulases have widespread applications the cost of production impede their
exploitation, particularly for cellulosic ethanol. Therefore, many laboratories are working on the
strategies to reduce the production cost by employing waste materials as media components for
enzyme production. Another area of research in focus will have novel immobilization matrices so
that the catalytic process can be efficient and ultimately the cost can be reduced. The recent
advancement in synthetic biology has opened a new arena for the development of chimeric
cellulases. Improvements in the above areas of research will lead toward a sustainable use of
resources, maximizing their use through cost-effective, low-energy and environment-friendly
green bioprocesses.

REFERENCES

1.Sharmila d, “Comparative Study on Production of Cellulase in Fresh and Sea Water by

Fursarium subglutinans MTCC 11891” Master of Technology in Biotechnology ,2014 ,20-28
2.Khushboo Pala, Tanu Sharma, Dr. Divya Sharma, “An Overview of Cellulase Production and
Industrial Applications” 2021,11-12
3.Sharmila d, “Comparative Study on Production of Cellulase in Fresh and Sea Water by
Fusarium subglutinans MTCC 11891” Master of Technology in Biotechnology ,2014 , 1-3
4.Khushboo Pala, Tanu Sharma, Dr. Divya Sharma, “An Overview of Cellulase Production and
Industrial Applications” 2021, 18- 26
5.Petar Keković, “Production and purification of the cellulase enzymatic complex of
Trichoderma reesei Rut-C30” 2016, 20.21,11-21
6.Jerin Isnat Abedin, “Isolation And Identification of cellulose degrading bacteria from soil
samples. “
2015, 3,4, 4-20
7.R.K.Sukumaran, R.R.Singhania, A. Pandey (2005), J. Sci. Ind, 64,832-844
8.R.Gupta, G.Mehta, D.Deswal, S.Sharma, K.K.Jain, A. Singh, R.C.Kuhad, in: R.C. Kuhad and
A.Singh (eds.) (2013).Biotechnology for Environmental Management and Resource Recovery,
89-106
9.Saini A., Aggarwal N.K., Yadav A. (2017).Cost-effective cellulase Production using
Parthenium hysterophorus biomass as an unconventional lignocellulosic substrate, 3 Biotech,
pp.7-12
10.Prasanna H.N., Ramanjaneyulu G., Rajasekhar R.B. (2016).Optimization of cellulase
production by Penicillium sp., 3 Biotech, 6, 162
11.M. K. Bhat (2000).Cellulase and related enzymes in biotechnology, Biotechnol. Adv.18, 355-
383
12. Uroosa ejaz, Md sohail(2021).Cellulases: From Bioactivity to a Variety of Industrial
Applications