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International Journal of Biochemistry & Biotech Science 2012; 1: 1-12 1
MICROBIAL CELLULASES AND ITS APPLICTIONS: A
REVIEW
P.Saranraj*, D. Stella and D. Reetha
* P.Saranraj
Department of Microbiology,
Annamalai University,
Annamalai Nagar,
Chidambaram – 608 002, E mail: [email protected]
Abstract
Numerous agricultural residues generated due to diverse agricultural practices and food
processing such as rice straw, yam peels, cassava peels, banana peels among others represents
one of the most important energy resources. The major components of these are cellulose and
hemicellulose (75-80%) while lignin constitutes only 14%. Yearly accumulation of these
agricultural residues causes deterioration of the environment and huge loss of potentially
valuable nutritional constituents which when processed could yield food, feed, fuel, chemicals
and minerals. Agricultural residues when dumped in open environment constitute health hazard
due to pollution and support for the growth of microorganisms. The present review is focused on
microbial cellulases and its applications. Cellulose is considered as one of the most important
sources of carbon on this planet and its annual biosynthesis by both land plants and marine algae
occurs in many tones per annum. Recycling of agricultural residue can be achieved naturally and
artificially by microorganisms. Aerobic organisms such as fungi, bacteria, and some anaerobic
organisms have been shown to be able to degrade some constituents of these residues. Fungi
play a significant role in the degradation of cellulose under aerobic conditions. Cellulases are
important enzymes not only for their potent applications in different industries, like industries of
food processing, animal feed production, pulp and paper production , and in detergent and textile
industry, but also for the significant role in bioconversion of agriculture wastes in to sugar and
bioethanol. This review assesses the following topics: cellulose in agricultural wastes, cellulases
and its types, cellulolytic microorganisms, microbial degradation of cellulose and cellulase
production, microbial fermentation for cellulase production and application of cellulases.
Keywords: Cellulose, Agricultural wastes, Cellulase, Microorganisms and Fermentation.
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International Journal of Biochemistry & Biotech Science 2012; 1: 1-12 2
Introduction
espite a worldwide and enormous utilization of natural cellulosic sources, there are still
abundant quantities of cellulose-containing raw materials and waste products that are not
exploited or which could be used more efficiently. The problem in this respect is however to
develop processes that are economically profitable. Cellulose containing wastes may be
agricultural, urban, or industrial in origin, sewage sludge might also be considered a source of
cellulose since its cellulosic content provides the carbon needed for methane production in
the anaerobic digestion of sludge [1]. Agricultural wastes include crop residue, animal
excreta and crop processing wastes slashing generated in logging, saw dust formed in timber
production and wood products in forestry originated activities
Cellulose is earth’s major biopolymer and is of tremendous economic importance around the
globe. Cellulose is the major constituent of raw materials like cotton (over 94%) and wood
(over 50%). Cellulose is the primary structural component of the plant cell wall. It accounts
for over half of the carbon in the biosphere. Approximately 1015 of cellulose were estimated
to be synthesized and degraded annually. Cellulose is predominantly of plant origin, it also
occurs in the stiff outer mantles of marine invertebrates known as tunicates (urochordates).
Cellulose from major land plants as forest trees and cotton is assembled from glucose, which
is produced in the living plant cell from photosynthesis. In the oceans, however, unicellular
plankton produces most cellulose or algae using the same type of carbon-di-oxide fixation
found in photosynthesis of land plants. It is estimated that the amount of carbon assimilated
by plants throughout the year is 200 billion tones. Plants in the form of structural
polysaccharides, which human beings cannot degrade, store most of this energy [2].
Cellulosic biomass offers a possible solution. It is a complex mixture of carbohydrate
polymers known as cellulose, hemicellulose, lignin, and a small amount of compounds
known as extractives. Examples of cellulosic biomass include agricultural and forestry
residues, municipal solid waste, herbaceous and woody plants, and underused standing
forests. Cellulose is composed of glucose molecules bonded together in long chains that form
a crystalline structure [3]. Cellulose is a fibrous, tough, water-insoluble substance.
Hemicellulose is not soluble in water. It is a mixture of polymers made up from xylose,
mannose, galactose, or arabinose. Hemicellulose is much less stable than cellulose. Lignin,
which is present along with cellulose in trees, is a complex aromatic polymer of
phenylpropane building blocks. Lignin is resistant to biological degradation [4].
Cellulose in agricultural wastes Agriculture wastes contain a high proportion of cellulosic matter which is easily decomposed
by a combination of physical, chemical and biological processes. The bunch consists of 70
moisture and 30% solid; of which holocellulose accounts for 65.5, lignin 21.2, ash 3.5, hot
water-soluble substances 5.6 and alcohol-benzene soluble 4-1% [5]. Lignin is an integral cell
wall constituent, which provides plant strength and resistance to microbial degradation [6].
The recognition that environmental pollution is a worldwide threat to public health has given
rise to a new massive industry for environmental restoration. Biological degradation, for both
economic and ecological reasons, has become an increasingly popular alternative for the
treatment of agricultural, industrial, organic as well as toxic waste. These wastes have been
insufficiently disposed leading to environmental pollution.
D
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Plant lignocellulosics as organic substances are subject to attacks by biological agents such
as fungi, bacteria and insects. Acids can breakdown the long chains in cellulose to release the
sugars through hydrolysis reaction, but because of their high specificity, cellulase can achieve
higher yield of glucose from cellulose. A portion of pretreated biomass can be used to feed a
fungus or other organism that produces cellulase that can then be added to pretreated solids to
release glucose from cellulose [7]. Filamentous fungi which use cellulose as carbon source
possess the unique ability to degrade cellulose molecules in plant lignocellulose. Although, a
large number of microorganisms are capable of degrading cellulose, only a few of these
produce significant quantities of cell-free enzymes capable of completely hydrolyzing
crystalline cellulose in vitro [8].
Cellulases
Bioconversion of cellulose containing raw materials is an important problem of current
biotechnology due to the increasing demand for energy, food and chemicals. Cellulases are
enzymes which hydrolyze the β-1,4- glycosidic linkage of cellulose and synthesized by
microorganisms during their growth on cellulosic materials [9]. The complete enzymatic
hydrolysis of cellulosic materials needs different types of cellulase; namely endoglucanase,
(1,4-D-glucan-4-glucanohydrolase; EC 3.2.1.4), exocellobiohydrolase (1, 4-D-glucan
glucohydrolase; EC 3.2.1.74) and glucosidase (D-glucoside glucohydrolase; EC 3.2.1.21).
Enzymatic process to hydrolyze cellulosic materials could be accomplished through a
complex reaction of these various enzymes. Two significant attributes of these enzyme-based
bioconversion technologies are reaction conditions and the production cost of the related
enzyme system. Therefore, worldwide there has been many research works focused on
obtaining new microorganisms producing celluloytic enzymes with higher 105 specific
activities and greater efficiency [10]. Enzymes produced by marine microorganisms can
provide numerous advantages over traditional enzymes due to the wide range of
environments [11].
Cellulases are comprised of independently folding, structurally and functionally discrete units
called domains or modules, making cellulases modular [12]. A typical free cellulase is
composed of a carbohydrate binding domain (CBD) at the C-terminal joined by a short poly-
linker region to the catalytic domain at the N-terminal. There are only two modes of action
for the hydrolysis of cellulose by cellulases, either inversion or retention of the configuration
of the anomeric carbon. At least two amino acids with carboxyl groups located within the
active site catalyze the reaction by acid-base catalysis.
The commonly described mode of action for cellulases on polymers is either exo- or endo-
cleavage, and all cellulases target the specific cleavage of β-1,4-glycosidic bonds. Using this
classification system, cellobiohydrolases (exoglucanases) were classified as exo-acting based
on the assumption that they all cleave β-1,4-glycosidic bonds from chain ends. As well, those
enzymes truly exo-acting often have a tunnel-shaped closed active site which retains a single
glucan chain and prevents it from readhering to the cellulose crystal [13]. While
endoglucanases on the other hand, are often classified as endo-acting cellulases because they
are thought to cleave β-1, 4-glycosidic bonds internally only and appear to have cleft-shaped
open active sites.
Endoglucanase are active on amorphous regions of cellulose and thus their activity can be
assayed using soluble cellulose substrates; i.e., the carboxymethylcellulase assay (CMCase).
However, there is now supporting evidence that some cellulases display both modes of
action, endo- and exo- [14]. Thus classification has changed; cellobiohydrolases
(exoglucanases) are described as active on the crystalline regions of cellulose; whereas,
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International Journal of Biochemistry & Biotech Science 2012; 1: 1-12 4
endoglucanases are typically active on the more soluble amorphous region of the cellulose
crystal. There is a high degree of synergy seen between cellobiohydrolases (exoglucanases)
and endoglucanases, and it is this synergy that is required for the efficient hydrolysis of
cellulose crystals.
Types of cellulases
Five general types of cellulases based on the type of reaction catalyzed:
1. Endo-cellulase breaks internal bonds to disrupt the crystalline structure of
cellulose and expose individual cellulose polysaccharide chains.
2. Exo-cellulase cleaves 2-4 units from the ends of the exposed chains produced by
endocellulase, resulting in the tetrasaccharides or disaccharide such as cellobiose.
3. There are two main types of exo-cellulases (or cellobiohydrolases, abbreviate
CBH) - one type working processively from the reducing end, and one type
working processively from the non-reducing end of cellulose.
4. Cellobiase or beta-glucosidase hydrolyses the exo-cellulase product into individual
monosaccharides.
5. Oxidative cellulases that depolymerize cellulose by radical reactions, as for
instance cellobiose dehydrogenase (acceptor).
Cellulose phosphorylases that depolymerize cellulose using phosphates instead of water. In
the most familiar case of cellulase activity, the enzyme complex breaks down cellulose to
beta-glucose. This type of cellulase is produced mainly by symbiotic bacteria in the
ruminating chambers of herbivores. Aside from ruminants, most animals (including humans)
do not produce cellulase in their bodies, and are therefore unable to use most of the energy
contained in plant material. Enzymes which hydrolyze hemicellulose are usually referred to
as hemicellulase and are usually classified under cellulase in general. Enzymes that cleave
lignin are occasionally classified as cellulase, but this is usually considered erroneous. Within
the above types, there are also progressive (also known as processive) and non-progressive
types. Progressive cellulase will continue to interact with a single polysaccharide strand; non-
progressive cellulase will interact once then disengage and engage another polysaccharide
strand. Most fungal cellulases have a two-domain structure with one catalytic domain, and
one cellulose binding domain, that are connected by a flexible linker. This structure is
adoption for working on an insoluble substrate and it allows the enzyme to diffuse two-
dimensionally on a surface in a caterpillar way. However, there are also cellulases (mostly
endoglucanases) that lacks cellulose binding domain. These enzymes might have a swelling
function.
Cellulolytic microorganisms A variety of microorganisms take part in Cellulose hydrolysis with an aid of a multienzyme
system. Among the best-characterized cellulase systems are as follows: White rot fungus
Phanerochaete chrysosporium, Soft-rot fungi, Fusarium solani, Penicillum funiculosum,
Talaromyces emersonii, Trichoderma koningii and Trichoderma reesei. Some of the
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International Journal of Biochemistry & Biotech Science 2012; 1: 1-12 5
aerobic cellulolytic bacteria which are having best-characterized cellulase systems are as
follows: Cellulomonas sp., Cellvibrio sp., Microbispora bispora and Thermomonospora sp.
Examples of anaerobic cellulolytic bacteria are as follows: Acetivibrio cellulolyticus,
Bacteroides cellulosolvens, Bacteriodes succinogenes, Clostridium thermocellum,
Ruminococcus albus and Ruminococcus flavefaciens. Fungi are the main cellulase producing
microorganisms, though a few bacteria and actinomycetes have also been reported to yield
cellulase activity. Fungal genera like Trichoderma and Aspergillus are known to be cellulase
producers and crude enzymes produced by these microorganisms are commercially available
for agricultural use. The genus Aspergillus species attack cellulose producing significant
amount of cell free cellulase capable of hydrolyzing cellulose into fermentable soluble sugars
such as glucose; an important raw material in chemical industries. Aspergillus and
Trichoderma specie are well known efficient producers of cellulases [15]. Several studies
have been carried out to produce cellulolytic enzymes from biowaste degradation process by
many microorganisms including fungi such as Trichoderma, Penicillium and Aspergillus
species etc., by Mandels and Reese [16].
Microbial degradation of cellulose and cellulase production
Microorganisms bring about most of the cellulose degradation occurring in nature. They meet
this challenge with the aid of a multi-enzyme system. They include fungi and bacteria,
aerobes and anaerobes, mesophiles and thermophiles and occupy a variety of habitats.
Aerobic bacteria produced numerous individual, extra-cellular enzymes with binding
modules for different cellulose conformations. Anaerobic bacteria possess a unique
extracellular multienzyme complex, called cellulosome. Binding to a non-catalytic structural
protein (scaffoldin) stimulates activity of the single components towards the crystalline
substrate. The most complex and best investigated cellulosome is that of the thermophilic
bacterium Clostridium thermocellum.
Cellulase preparations are able to decompose natural cellulose (e.g. filter paper) as well as
modified celluloses such as carboxymethyl cellulose or hydroxyethyl cellulose.
Cellulasehydrolyses 1,4-β-D-glucosidic linkages in cellulose, licheninand cereal β -D-
glucans. The exoglucanases are thought to act primarily on newly generated chain ends
producing mainlycellobiose , β-Glucosidase hydrolyses terminal β-D-glucose residues from
the ends of cellulose molecules. In nature, cellulose is found in association with other
components e.g. hemicellulose, lignin and pectin. SERVA cellulases contain a number of
other activities, which assist in breaking down these components and degrading cell walls. α-
Amylase hydrolyses 1,4- α -D-glucosidic linkages in polysaccharides containing three or
more 1,4- α -linked D-glucose units. Pectinase randomly cleaves 1, 4- α -D-galactosiduronic
linkages in galacturans. These products also contain hemicellulase and protease activities.
Cellulase is used to modify the surface properties of cellulosic fibers and fabric in order to
achieve a desired surface effect [17]. Cellulase has been used to degrade environmental
wastes such as plant wastes (lignocellulosics). Cellulase as an industrial enzyme is imported
for use in Nigeria. Therefore, its production using readily available sources (example plant
residues) will help reduce importation costs. It is against this background, that this study was
carried out to evaluate the cellulase activity of Aspergillus candidus on various agro-forestry
residues as feed substrates and to determine the effects of pH on cellulase activity. Cellulase
production by different organisms in submerged state fermentation has received more
attention and is found to be cost-prohibitive because of high cost of process engineering.
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Microbial fermentation for cellulase production.
Many microorganisms are capable of degrading and utilizing cellulose and hemicellulose as
carbon and energy sources. During composting, the capacity of thermophilic microorganisms
to assimilate organic matter depends on their ability to produce the enzymes needed for
degradation of the substrate [18]. Enzymatic hydrolysis processing of cellulosic materials
could be accomplished through a complex reaction of various enzymes. Cellulases are
inducible enzymes which are synthesized by microorganisms during their growth on
cellulosic materials [19]. Both fungi and bacteria have been heavily exploited for their
abilities to produce a wide variety of cellulases and hemicellulases. Most emphasis has been
placed on the use of fungi because of their capability to produce copious amounts of
cellulases and hemicellulases which are secreted to the medium for easy extraction and
purification. In addition, the 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 host such as Escherichia coli.
Most importantly, bacteria inhabit a wide variety of environmental and industrial niches,
which produce cellulolytic strains that are extremely resistant to environmental stresses.
These include strains that are thermophilic or psychrophilic, alkaliphilic or acidiophilic, and,
strains that are halophilic. Not only can these strains survive the harsh conditions found in the
bioconversion process, but they often produce enzymes that are stable under extreme
conditions which may be present in the bioconversion process and this may increase rates of
enzymatic hydrolysis, fermentation, and, product recovery. Researchers are now focusing on
utilizing, and improving these enzymes for use in the biofuel and bioproduct industries.
Cellulose, being an abundant and renewable resource, is a potential raw material for the
microbial production of food, fuel and chemicals. Various bacteria, actinomycetes and
filamentous fungi produce extra cellular cellulases when grown on cellulosic substrates
though many actinomycetes have been reported to have less cellulase activity than moulds.
Investigations on the extracellular cellulases of fungi have been concentrated mainly on
Trichoderma sp. and studies on other mesophilic fungi suggested the possibility that other
cellulase systems could be utilized for the hydrolysis of cellulose [20].
Maulin Shah et al. investigated the ability Phylosticta sp. and Aspergillus sp. to produce
various lignolytic and cellulolytic enzymes such as laccase, lignin peroxidase, xylanase,
endo-1,4-β-d-glucanase (CMCase) and exo-1,4-β-d-glucanase [filter paper activity (FP
activity)] on banana agricultural waste (leaf and pseudostem biomass) biomass under solid
state fermentation (SSF) condition [21]. The production pattern of these enzymes was studied
during the growth on the organisms for a period of 40 days. Very low levels of cellulolytic
enzyme activities were observed compared to lignin degrading enzymes by both the
organisms. Maximum specific activities of studied enzymes were obtained at 20 days of
culture growth.
Narasimha et al. compared the production of cellulase (filter paper activity, endoglucanase
and (glucosidase) by Aspergillus niger on three media in liquid shake culture [22]. The
culture filtrate of this organism exhibited relatively highest activity of all three enzymes and
extracellular protein content at 7 days interval during the course of its growth on Czapek-Dox
medium supplemented with 1.0% (w/v) cellulose. Urea as a nitrogen source and pH 5.0 were
found to be optimal for growth and cellulase production by Aspergillus niger. Among various
soluble organic carbon sources and lignocelluloses tested, carboxymethylcellulose and
sawdust at 1% supported maximum production of all three enzymes by Aspergillus niger.
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International Journal of Biochemistry & Biotech Science 2012; 1: 1-12 7
Reeta Rani Singhana et al. carried out cellulase production studies using the fungal culture
Trichoderma reesei using four different lignocellulosic residues (both raw and pre-treated) by
solid-state fermentation [23]. The effect of basic fermentation parameters on enzyme
production was studied. Maximal cellulase production obtained was 154.58 U/gds when pre-
treated sugarcane bagasse (PSCB) was used as substrate. The optimal conditions for cellulase
production using PSCB were found to be initial moisture content - 66%, initial medium pH-
7.0, incubation temperature -28°C, NH4NO3 at 0.075 M, and 0.005 M cellobiose. The
optimal incubation time for production was 72 hrs. Results indicate the scope for further
optimization of the production conditions to obtain higher cellulase titres using the strain
under SSF.
Munir khan et al. carried out cellulase production by solid state bioconversion (SSB) method
using rice straw, a lignocellulosic material and agricultural waste, as the substrate of three
Trichoderma sp. and Phanerochaete chrysosporium in lab-scale experiments [24]. The results
were compared to select the best fungi among them for the production of cellulase.
Phanerochaete chrysosporium was found to be the best among these species of fungi, which
produced the highest cellulase enzyme of 1.43 IU/mL of filter paper activity (FPase) and
2.40 IU/mL of carboxymethylcellulose activity (CMCase). The “glucosamine” and “reducing
sugar” parameters were observed to evaluate the growth and substrate utilization in the
experiments. In the case of Phanerochaete chrysosporium, the highest glucosamine
concentration was 1.60 g/L and a high concentration of the release of reducing sugar was
measured as 2.58 g/L obtained on the 4th day of fermentation.
Acharya et al. focused the factors relevant for improvement of enzymatic hydrolysis of saw
dust by using Aspergillus niger. Different cultural conditions were examined to assess their
effect in optimizing enzyme production [25]. Alkaline pretreated (2 N NaOH) saw dust at
9.6% concentration gave 0.1813 IU/mL cellulase activity. Optimum pH for cellulase
production was between 4.0 and 4.5. Submerged fermentation at 120 rpm at 28°C gave
higher yields of cellulase compared to static condition. Several other parameters like
inoculum size, time duration, nitrogen source and its concentration were also optimized for
the cellulase production by using saw dust as substrate.
Sherif et al. isolated twelve Aspergillus species from some local soil samples [26]. On the
basis of cellulolytic activity, Aspergillus fumigatus was selected and used for production of
exoglucanase , endoglucanase , CMCase, β-glucosidase and xylanase by adopting SSF
condition using mixed substrate of rice straw amended with wheat bran. Effect of Culture
conditions including; incubation period, initial pH, incubation temperature, moisture level,
different nitrogen sources, different lignocelluloses as carbon source and different ratios of
mixed rice straw and wheat bran were evaluated. The fungus expressed high enzyme
production after 4 days incubation at moisture level 75%, initial pH 5-6, at 40°C in presence
of NaNO3 as an inorganic nitrogen source. The recorded activities were 14.71, 8.51, 0.93,
0.68 and 42.7 IU g-1 for CMCase, β-glucosidase, exoglucanase, endoglucanase and xylanase,
respectively.
Milila et al. used rice husk, millet straw, guinea corn stalk and sawdust as fermentation feed
substrate for the evaluation of cellulase activity secreted by Aspergillus candidus [27]. The
substrates were pretreated with 5% NaOH (alkaline treatment) and autoclaved. From the
fermentation studies, rice husk, millet straw and guinea corn stalk feed substrates showed the
highest cellulase activity of 7.50, 6.88 and 5.84 IU, respectively. The effect of pH showed
that optimal pH for maximum cellulase activity varied in each of the substrates used. Rice
husk and millet straw had maximum enzyme activity at pH 5, while guinea corn stalk and
sawdust had maximum activity at pH 3 and 4, respectively.
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International Journal of Biochemistry & Biotech Science 2012; 1: 1-12 8
Abo-State et al. isolates twenty nine fungal strains from agriculture wastes [28]. Aspergillus
sp. was the predominant genera in these agriculture wastes. The most potent cellulase
producers were selected for studying their cellulase productivities on Wheat Straw (WS),
Wheat Bran (WB), Rice Straw (RS) and Corn Cob (CC) as cheap, renewable agriculture
wastes by solid state fermentation (SSF). Five Aspergillus sp. and standard strain
Trichoderma viride were grown on the agriculture wastes and CMCase, FPase, Avicelase and
soluble protein were determined. Trichoderma viride produces the highest CMCase on WS
(555U/ml), while the highest FPase (141U/ml) and Avicelase (46U/ml) were produced on
WB. The isolated strain Aspergillus MAM-F35 gave the highest CMCase (487U/ml), FPase
(79U/ml) and Avicelase (35U/ml) on WS.
Fatma et al. production of cellulase by Trichoderma reesei cultivated on alkali treated rice
straw using solid state fermentation (SSF) technique [29]. The high cellulase activity was
obtained when the fungus was cultivated on substrate with about 75 % (v/w) moisture, pH 4.8
for 5 days incubation at 28 ± 2ºC, as it gave 16.2 IU/g substrate. The obtained cellulase of 1.2
IU/ ml culture filtrate was applied for saccharification (5% w/v) of alkali treated rice straw, in
0.1M citrate buffer pH 4.8 in shaker water bath of 100 rpm. Sugary solution of 1.07 %
glucose was achieved after 16 hrs. The sugary solution was concentrated to give 10% (w/v)
glucose. Ethanolic fermentation was conducted by Saccharomyces cerevisiae under static
condition giving 5.1% (v/v) ethanol after 24 hrs. The fermented mash contained 3.6 g/L yeast
cell can be utilized as fooder yeast used for animal feeding.
Narmeen El Sersy et al. screened six marine strains of Actinomycetes for their carboxymethyl
cellulase (CMCase) productivity [30]. Streptomyces ruber was chosen to be the best
producing strain. The highest enzyme production (25.6 U/ml) was detected at pH 6 and 40°C
after 7 days of incubation. Plackett-Burman design was applied to optimize the different
culture conditions affecting enzyme production. Results showed that a high concentration of
KH2PO4, and a low concentration of MgSO4 had a significant effect on enzyme production.
Rice straw was used as a low cost source of cellulose. It was found that 30 g/l rice straw was
the suitable concentration for maximum enzyme production. Partial purification of cellulase
enzyme using an anion exchange chromatography resulted in the detection of two different
types of CMCases, type I and II, with specific activity of 4239.697 and 846.752 U/mg,
respectively.
Hafiz Iqbal et al. investigated the potential of a filamentous fungus, Trichoderma harzianum
for hyper-production of third most demanded industrial enzyme carboxymethyl cellulase
using cheap and easily available agro-industrial residue wheat straw as growth supporting
substrate under still culture solid state fermentation technique [31]. Production of
carboxymethyl cellulase was substantially enhanced through media optimization process. To
promote carboxymethyl cellulase production, they evaluated the effect of several kinetic
parameters like pretreatment, substrate concentration, initial moisture content, pH, incubation
temperature and inoculum size on carboxymethyl cellulase production. Samples were
harvested after every 24 hrs to study the profile of cellulase enzyme produced by the fungus
on proximally analyzed wheat straw. By optimizing the SSF medium containing 2 % HCl
pretreated wheat straw; maximum carboxymethyl cellulase activity (480±4.22 μM /mL/min)
was recorded after 7th day of incubation at pH 5.5; temperature, 35°C; moisture, 40 % and
inoculum size, 10 %, using optimum substrate concentration (3%).
Siva Sakthi et al. isolated Aspergillus niger from the spoiled coconut and identified using
LPCB staining based on its morphological and cultural features [32]. Optimization of
cellulase production was done by using various physical (Temperature, pH, Salinity and
Incubation time) and chemical parameters (Carbon sources and Nitrogen sources) which
could influence the enzyme activity. Cellulase production was maximum at the temperature
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International Journal of Biochemistry & Biotech Science 2012; 1: 1-12 9
20°C and minimum at 40ºC. The optimal pH for the cellulase production was observed
maximum in 6.0 and minimum in 7.0. Cellulase production was maximum at 48 hrs and
minimum at 24 hrs. Cellulase production was maximum with when fructose was used as a
carbon source and minimum with sucrose. Cellulase production was maximum when Malt
extract was used as a nitrogen source and minimum with yeast extract.
Applications of cellulases
Cellulases were initially investigated several decades back for the bioconversion of
biomass which gave way to research in the industrial applications of the enzyme in
animal feed, food, textiles and detergents and in the paper industry. With the shortage
of fossil fuels and the arising need to find alternative source for renewable energy
and fuels, there is a renewal of interest in the bioconversion of lignocellulosic biomass
using cellulases and other enzymes. In the other fields, however, the technologies and
products using cellulases have reached the stage where these enzymes have become
indispensable.
Textile industry
Cellulases have become the third largest group of enzymes used in the industry since
their introduction only since a decade. They are used in the bio- stoning of denim
garments for producing softness and the faded look of denim garments replacing the use of
pumice stones which were traditionally employed in the industry. They act on the
cellulose fiber to release the indigo dye used for coloring the fabric producing the faded
look of denim. Humicola insolens cellulase is most commonly employed in the equally
good cellulases are utilized for digesting off the small fiber ends protruding from the
fabric resulting in a better finish cellulases, used in softening defibrillation , and in
processes for providing localized variation in the color density of fibers.
Laundry and detergent
Cellulases, in particular EG III and CBH I, are commonly used in detergents for
cleaning textiles Several reports disclose that EG III variants, in particular from
Trichoderma reesei are suitable for the use in detergents. Trichoderma viride and
Trichoderma harzianum are also industrially utilized natural sources of cellulases, as
Aspergillus niger. Cellulase preparations, mainly from species of Humicola (Humicola
insolens and Humicola griseathermoidea) that are active under mild alkaline conditions
and at elevated temperatures, are commonly added in washing powders , and in
detergents.
Food and animal feed
In food industry, cellulases are used in extraction and clarification of fruit and
vegetable juices. production of fruit nectars and purees, and in the extraction of olive
oil Glucanases are added to improve the malting of barley in beer manufacturing and in
wine industry, better maceration and color extraction is achieved by use of exogenous
hemicellulases and glucanases. Cellulases are also used in carotenoid extraction in the
production of food coloring agents.
Enzyme preparations containing hemicellulase and pectinase in addition to cellulases are
used to improve the nutritive quality of forages. Improvements in feed digestibility and
animal performance are reported with the use of cellulases in feed processing describes
the feed additive use of Trichoderma cellulases in improving the feed conversion ratio
and increasing the digestibility of a cereal-based feed.
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International Journal of Biochemistry & Biotech Science 2012; 1: 1-12 10
Pulp and paper industry
In the pulp and paper industry, cellulases and hemicellulases have been employed for
biomechanical pulping for modification of the coarse mechanical pulp and hand sheet
strength properties de-inking of recycled fibers and for improving drainage and run ability
of paper mills. Cellulases are employed in the removing of inks coating and toners
from paper Bio characterization of pulp fibers is another application where microbial
cellulases are employed. Cellulases are also used in preparation of easily biodegradable
cardboard. The enzyme is employed in the manufacture of soft paper including paper
towels and sanitary paper and preparations containing cellulases are used to remove
adhered paper.
Biofuel
Perhaps the most important application currently being investigated actively is in the
utilization of lignocellulosic wastes for the production of biofuel. The lignocellulosic
residues represent the most abundant renewable resource available to mankind but their
use is limited only due to lack of cost effective technologies. A potential application
of cellulase is the conversion of cellulosic materials to glucose and other fermentable
sugars, which in turn can be, used as microbial substrates for the production of single cell
proteins or a variety of fermentation products like ethanol.
Organisms with cellulose systems that are capable of converting biomass to alcohol
directly are already reported. But, none of these systems described are effective alone to
yield a commercially viable process. The strategy employed currently in bioethanol
production from lignocellulosic residues is a multi-step process involving pre-treatment
of the residue to remove lignin and hemicellulase fraction, cellulase treatment at 50°C to
hydrolyze the cellulosic residue to generate fermentable sugars, and finally use of a
fermentative microorganism to produce alcohol from the hydrolyzed cellulosic material.
The cellulose preparation needed for the bioethanol plant is prepared in the premises
using same lignocellulosic residue as substrate, and the organism employed is almost
always Trichoderma ressei. To develop efficient technologies for biofuel production,
significant research has been directed towards the identification of efficient cellulase
systems and process conditions besides studies directed at the biochemical and genetic
improvement of the existing organisms utilized in the process. The use of pure enzymes in
the conversion of biomass to ethanol or to fermentation products is currently
uneconomical due to the high cost of commercial cellulases.
Effective strategies are yet to resolve and active research has to be taken up in this direction.
Overall, cellulosic biomass is an attractive resource that can serve as substrate for the
production of value added metabolites and cellulases as such. Apart from these common
applications, cellulases are also employed in formulations for removal of industrial
slime , in research for generation of protoplast and for generation of antibacterial
chitooligosaccharides, which could be used in food preservation, immune modulation
and as a potent antitumor agent.
Conclusion
In the recent years, one of the most important biotechnological applications is the conversion
of agricultural wastes and all lignocellulosics into products of commercial interest such as
ethanol, glucose and single cell products. The key element in bioconversion process of
P.Saranraj et al.
International Journal of Biochemistry & Biotech Science 2012; 1: 1-12 11
lignocellulosics to these useful products is the hydrolytic enzymes mainly cellulases. The
bioconversions of cellulosic materials are now a subject of intensive research as a
contribution to the development of a large scale conversion process beneficial to mankind.
Such process would help alleviate shortages of food and animal feeds, solve modern waste
disposal problem and diminish man’s dependence on fossil fuels by providing a convenient
and renewable source of energy in the form of glucose. A diverse spectrum of cellulolytic
microorganism mainly fungi and bacteria have been isolated and identified over the years and
this still continue to grow rapidly. Fungi are the main cellulase producing microorganism and
Aspergillus and Trichoderma are the main fungal genera that were used for commercial
production of cellulase. Therefore the present review showing the ability of microorganisms
to synthesize high amount of extra cellular exoglucanase within a relatively short period of
time, utilizing agro wastes that would otherwise cause environmental pollution, could be used
for rapid and commercial production of cellulase
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