cellulase for biofuel-a review

Abstract

cellulases enzyme

Full text

International Journal of Recent Trends in Science And Technology, P-ISSN 2277-2812 E-ISSN 2249-8109
Special Issue, ACAEE: 2018 pp 17-25
Copyright © 2018, Statperson Publishing Corporation, International Journal of Recent Trends in Science And Technology, P-ISSN 2277-2812 E-ISSN 2249-8109, Special Issue, ACAEE: 2018
Original Research Article
Cellulases for biofuel: A review
Ashiq Magrey1*, Sanjay Sahay2, Ragini Gothalwal1
1Department of Biotechnology, Barkatullah University, Bhopal, Madhya Pradesh, INDIA.
2NSCB Government P.G College, Biaora, Rajgarh, Madhya Pradesh, INDIA.
Email: ashiqmagrey@gmail.com
Abstract Cellulases have wide applications in the biofuel industry. The three main components, namely endoglucanase,
exoglucanase, and β-glucosidase effectively convert lignocellulosic biomass into fermentable sugar. The commercial
production of cellulase is done using the submerged fermentation; however, it is costly and less economic for biofuels
production. Moreover, microbial cellulase production process suffers from various bottlenecks. Because of the low cost,
production of cellulase using solid-state fermentation by fungi is preferable. Cellulose is the main polymer in biomass
and cellulases can hydrolyze it to cellobiose, which can be converted to glucose by β-glucosidase. Extensive research is
being carried out to try to obtain cellulases with higher activity on pretreated biomass substrates by screening and
sequencing new organisms, engineering cellulases with improved properties and by identifying proteins that can
stimutate cellulases.. Despite extensive research on cellulases there are major gaps in our understanding of how they
hydrolyze crystalline cellulose, act synergistically, and the role of carbohydrate binding modules. Therefore, the present
review provides an overview on cellulase, main aspects of cellulase production and cellulase engineering for biofuels
industry.
Key Words: Biofuels production, cellulase, commercial cellulase, fungal cellulase, solid-statefermentation, submerged
fermentation,cellulase engineering.
*Address for Correspondence:
Ashiq Magrey, Department of Biotechnology, Barkatullah University, Bhopal, Madhya Pradesh, INDIA.
Email: ashiqmagrey@gmail.com
INTRODUCTION
Cellulase Enzymes: Enzymes are among the most
important products obtained for human needs through
Microbial sources. A large number of industrial processes
in the areas of industrial, environmental and food
biotechnology utilize enzymes at some stage or other.
Current developments in biotechnology are yielding new
applications for enzymes (Pandey et al., 1999). In the
present techno- economic era, procurance of energy is
one of the major problems which humanity is facing. All
the waste cellulose is a source of food and is also a
potential source of energy (Elder et al., 1986). Mandels et
al., (1974) reported that the breakdown of cellulose into
sugar can be achieved by acid hydrolysis as well as by
enzymatic hydrolysis. But enzymatic hydrolysis is mostly
preferred because it produces fewer by-products and
proceeds under milder condition. Cellulases have been
used and studied for most of the 20th century and are the
most commercially important of all the enzyme families.
The enzyme production is good by most of the fungi like
Aspergillus and Trichoderma Sp. It has been reported that
the structural complexity and rigidity of cellulosic
substrates have given rise to a remarkable divergence in
cellulose degradative enzymes. Microorganisms involved
in degrading these complex structures have faced many
evolutionary challenges, developing complex enzyme
systems to handle these varying substrates. Organisms
usually produce complex extracellular or membrane
bound cellulolytic enzymes comprising a combination of
activities (Mai et al., 2004). Klyosov (1995) found that
Cellulases are the group of hydrolytic enzymes that are
capable of hydrolyzing insoluble cellulose to produce
soluble oligosaccharides. Cellulases are modular enzymes
that are composed of independently folding, structurally
and functionally discrete units called domains. It has been
reported that there have been a notable differences found
in the cellulolytic enzymes isolated from various sources.
The differences were mainly in their polypeptide
Access this article online
Quick Response Code:
Website:
www.statperson.com
Accessed Date:
26 March 2018

Ashiq Magrey, Sanjay Sahay, Ragini Gothalwal
International Journal of Recent Trends in Science And Technology, P-ISSN 2277-2812 E-ISSN 2249-8109, Special Issue, ACAEE: 2018 Page 18
characteristics such as capacity to adsorb to cellulose,
molecular weight, isoelectric points, carbohydrate
content, catalytic activity, substrate specificity and amino
acid composition and sequence. However, a cellulase
system is an efficient hydrolysis of cellulose in a specific
coordinated manner. It is a combination of three
representative enzymes with or without cellulose binding
domains (Lynd et al., 2002). It has been seen that
cellulases are grouped along with hemicellulases and
other polysaccharide degrading enzymes such as
glycosidehydrolases and with the other auxiliary enzymes
including hemicelluloses hydrolases (Lynd et al., 2002).
But with the exponentially increasing genomic data, an
alternative classification based on amino acid sequence
similarities, of their catalytic domains has been suggested
( Lynd et al., 2002). While this system of classification
was very effective, it was unable to easily accommodate
enzymes that displayed both modes of catalysis, devising
further classification towards more conclusive structural
and mechanistic properties is yet required (Mai et al.,
2004).Cellulases are composed of independently folding,
structurally and functionally discrete units called domains
or modules, making cellulases module has been reported
by (Henrisat et al.,1998). Cellulases are inducible
enzymes synthesized by a large diversity of
microorganisms including both fungi and bacteria during
their growth on cellulosic materials (Kubicek et al., 1993;
Sanget et al., 2001). These microorganisms can be
aerobic, anaerobic, mesophilic or thermophilic. Among
them, the genera of Clostridium, Cellulomonas,
Thermomonospora, Trichoderma, and Aspergillus are the
most extensively studied cellulose producer (Khud et al.,
1999). Structurally fungal cellulases are simpler as
compared to bacterial cellulase systems, cellulosomes
(Bayer et al., 1994). Fungal cellulases typically have two
separate domains: a catalytic domain (CD) and a cellulose
binding module (CBM), which is joined by a short
polylinker region to the catalytic domain at the N-
terminal. The CBM is comprised of approximately 35
amino acids, and the linker region is rich in serine and
threonine. The main difference between cellulosomes and
free cellulase enzyme is in the component of
cellulosomes-cohesin containing scaffolding and dockerin
containing enzyme. The free cellulase contains cellulose
binding domains (CBMs), which are replaced by a
dockerin in cellulosomal complex, and a single
scaffolding-born CBM directs the entire cellulosomes
complex to cellulosic biomass (Carval et al., 2003; Bayer
et al.,2004).
Basic research on cellulases: (Wilson 2009 ) reported
that there has been extensive research on cellulases since
the end of World War II, there are still some major gaps
in our understanding of the mechanism by which they
catalyze the hydrolysis of crystalline cellulose. One gap is
information on the mechanism by which a cellulase binds
a segment of a cellulose chain from a microfibril into its
active site. This is probably the rate limit in gstep for
crystalline cellulose degradation, so that understanding
the mechanism of this step is very important for trying to
engineer cellulases with higher activity on real cellulose
substrates. Another gap is our understanding of how
cellulosomes are able to efficiently catalyze the
hydrolysis of cellulose, despite their large size that
restricts their ability to access much of the cellulose
surface area that is available to smaller free cellulases. A
third gap is an understanding of the way in which certain
free cellulose binding modules (CBM) stimulate cellulase
hydrolysis (Wang et al 2008. , and Moser et al., 2008 ). It
is possible that these domains modify the cellulose but
exactly how is not known. Finally, while there are some
plausible mechanisms for cellulase synergism, there is
still much more to be learned about this important process
(jeoh et al., 2006), particularly how mixtures of cellulases
hydrolyze both crystalline and amorphous regions in
bacterial cellulose while most individual enzymes only
seem to degrade amorphous regions (chen et al., 2007).
Classification of cellulases: Cellulase is a complex
enzyme system comprising of endo-1,4-β-D- glucanase
(endoglucanase, EC 3.2.1.4), exo-1,4-β-D-glucanase
(exoglucanase, EC 3.2.1.91) and β-D-glucosidase (β-D-
glucoside glucanhydrolase, EC 3.2.1.21) (Joshi and
panday, 1999).
Endoglucanase: Endoglucanase (endo-β-1,4-D-
glucanase, endo-β-1,4-D-glucan-4- glucano-hydrolase) -
often called as CMCase – hydrolyses carboxymethyl
cellulose (CMC) or swollen cellulose in a random
fashion. Accordingly, the length of the polymer
decreases, resulting in the increase of reducing sugar
concentration (Robson and chimbless, 1989 ; Begum et
al., 2009). Endoglucanase also acts on cellodextrins - the
intermediate product of cellulose hydrolysis-and converts
them to cellobiose (disaccharide) and glucose. These
enzymes are inactive against crystalline celluloses such as
cotton or avicel.
Exoglucanase: Exoglucanase (exo-β-1,4-D glucanase,
cellobiohydrolase) degrades cellulose by splitting-off the
cellobiose units from the non-reducing end of the chain. It
is also active against swollen, partially degraded
amorphous substrates and cellodextrins, but does not
hydrolyze soluble derivatives of cellulose like
carboxymethyl cellulose and hydroxyethyl cellulose.
Some cellulase systems also contain glucohydrolase (exo-
1,4-D-glucan-4-glucohydrolase) as a minor component
(Joshi and panday, 1999)..
β- glucosidase: β-glucosidase completes the process of
hydrolysis of cellulose by cleaving cellobiose and

International Journal of Recent Trends in Science And Technology, P-ISSN 2277-2812 E-ISSN 2249-8109
Special Issue, ACAEE: 2018 pp 17-25
Copyright © 2018, Statperson Publishing Corporation, International Journal of Recent Trends in Science And Technology, P-ISSN 2277-2812 E-ISSN 2249-8109, Special Issue, ACAEE: 2018
removing glucose from the non-reducing end (i.e., with a
free hydroxyl group at C-4) of oligosaccharides. The
enzyme also hydrolyzes alkyl and aryl β-glucosides
(Kubicek et al 1993).
Breakdown of Cellulose by Cellulases: Hydrolysis of
cellulose by the enzyme cellulase involves hydrolysis of
the glycosidic bonds connecting the β-D-glucosyl
residues of the cellulose. The general architecture of
cellulases features two discrete globular domains: a
catalytic domain, accountable for the hydrolysis reaction
itself and a cellulose-binding domain, with no catalytic
activity, nevertheless enhancing adsorption of the enzyme
on to insoluble macromolecular cellulose. In the native
enzymes the two domains are connected together by a
linker peptide (Sukumaran et al., 2005). Mechanistically,
cellulase is a family of 3 groups of enzymes, endo- (1,4)-
β-D-glucanase (EC 3.2.1.4), exo-(1,4)- β-D-glucanase
(EC 3.2.1.91) and β-glucosidases (EC 3.2.1.21). The
exoglucanase (CBH) acts on the terminals of the cellulose
chain and releases β-cellobiose as the end product;
endoglucanase (EG) randomly attacks the internal O-
glycosidic bonds producing glucan chains of different
lengths and the β-glycosidases act specifically on the β-
cellobiose disaccharides and produces glucose units.
Although the mechanism of cellulose degradation by
aerobic bacteria is similar to that of aerobic fungi but
anaerobic bacteria operate on different system.
Cellulosomes located on the cell surface mediate
adherence of anaerobic cellulolytic bacterial cells to the
substrate, which thereafter undergo a supramolecular
reorganization, so that the cellulosomal subunits
redistribute to interact with the various target substrates
(Kuhad et al., 2011 and Sukumaran et al., 2005).
Lignocellulosic ethanol production: Most plant material
is not sugar or starch, but contains cellulose,
hemicellulose and lignin. The cell wall of plant consists
of these three main substances. Hemicellulose and
cellulose-glucose chains are stacked each other into
crystalline fibrils, acting as a protector wall impenetrable
to water or enzymes. Lignin, a more complex
macromolecule, makes up of the rest (Schubert, 2006).
Cellulose and hemicellulose can be converted into ethanol
after converting them into sugar first, but lignin cannot.
The process is more complicated than converting starch
into sugar and then to ethanol. The first generation
technologies for biofuel production were based on
fermentation and distillation from sugar and starch rich
crops. The second generation technology for biofuel is
converting cellulose and hemicellulose from residues
such as straw, forestry brash and dedicated energy crops
to sugar, and then converted to biofuel by conventional
fermentation and distillation. The conversion of cellulosic
biomass (corn stove, wood chips) has a far higher
potential for fuel production than any of the conventional
biofuels. The challenge is biochemical plant lignins
incclude the cellulose cell walls and they must be
removed. Sugar in cellulosic biomass is locked up in
forms of cellulose and hemicellulose. Current
lignocellulosic ethanol technology are on three-stage
process; in the first stage lignocellulosic materials are
pretreated to lose the hemicelluloses-lignin bond and
increase the accessibility of water or enzyme to cellulose,
in the second stage cellulose is hydrolysed to glucose and
in the final stage glucose is put to fermentation. There are
many techniques for the pretreatment of lignocellulosic
biomass such as steam explosion, dilute acid treatment,
alkali treatment, ammonia fiber treatment etc. Among
them dilute acid pretreatment is preferred since it
removes a large fraction of the xylan and opens pores for
subsequent enzymatic hydrolysis of the cellulose
(Sannigrahi et al., 2011). For, hydrolysis of cellulose to
glucose there are two techniques viz., steam explosion at
high temperature/pressure and enzymatic one. Of these
two techniques, enzymatic is given more focus as the
other one is more energy intensive. The enzyme used for
hydrolysis is cellulases which is a composite enzyme
whose three main components together to bring about
complete hydrolysis of cellulose as follows
a) Exo- β-1-4, glucanase: It acts on the non
reducing end of the cellulose chain and
successively removes single glucose units
b) Endo-β-1-4, glucanase: It randomly attacks the
internal β-1-4, linkages
c) β-glucosidases or Cellobiases: It eventually
breaks down cellobiose, the building unit of
cellulose, to glucose.
Three areas were focused upon in current research to
bring down costs and increase productivity: developing
energy crops dedicated to biofuel production, improving
enzymes that deconstruct cellulosic biomass and
optimising microbes for industrial scale conversion for
biomass sugars into ethanol and other biofuel or bio
products.
Cellulases for Biofuels Production: Depletion of fossil
fuels and the increasing demand of alternate sources for
renewable energy have developed a huge interest in
cellulase production (Pandey et al., 2012). Cellulases
have
potential application in biofuels production.
Bioconversion of lignocellulosic substrate using
cellulases and other enzymes are the thirst area for the
commercialization of biofuels. The cellulase preparation
in biomass conversion processes is based on number of its
properties such as stability, product inhibition, synergism,
and composition of lignocellulosic biomass etc.
(Srivastava et al., 2015b). Commercial cellulases are

Ashiq Magrey, Sanjay Sahay, Ragini Gothalwal
International Journal of Recent Trends in Science And Technology, P-ISSN 2277-2812 E-ISSN 2249-8109, Special Issue, ACAEE: 2018 Page 20
found in the market by different names for biomass
hydrolysis. Nieves et al. (2009) have analyzed various
commercial cellulases for the hydrolysis of
biomass.These authors performed the standard enzymes
assays of Filter Paper Activity (FPU), CM Case (EG), β-
glucosidase (BGL) as well as xylanase. However, no
clear relation between the enzymatic activities of
cellulase on soluble and insoluble substrates could be
found. Therefore, it is unpredictable to explain the
efficiency of cellulase for effective bioconversion of
cellulosic biomass. Nevertheless, cellulases having higher
FPUs are desirable for effective conversion of biomass.
Cellulase enzymes can be used to convert the cellulose
portion of non food biomass, such as agricultural waste
and energy crops, into fermentable sugars for subsequent
conversion to renewable fuels and chemicals. Reprinted
(adapted) with permission from (Payne et al., 2015).
ADVANCED DEVELOPMENT IN
CELLULASES PRODUCTION PROCESSES
Production of cellulase is the thirst area of research
around the world for cost-effective production of biofuels
and, therefore,many researchers are consistently working
in this field. Low production and high cost have always
been a major constrain which are needed to be overcome
by adopting the novel and versatile approaches. In this
context, use of inexpensive raw materials as the
substrates, use of genetically modified microorganisms,
use of efficient crude thermostable/thermophilic enzyme
are some of the key factors which can enhance the
cellulase production, significantly (Srivastava et al.,
2015a). Srivastava et al. (2015a) have discussed the use
of thermostable enzyme using variousmicroorganisms for
partial/complete hydrolysis of cellulose. These authors
have systematically discussed the utility of thermostable
cellulase over cellulase for effective hydrolysis. Ang et
al. (2013) reported thermostable cellulase production
from Aspergillus fumigatus SK1. These thermostable
cellulose exhibited higher FPU activity with effective
hydrolysis for shorter time period. It has also been
reported that the thermostable enzyme can reduce the
hydrolysis time (Dutta et al., 2014; Srivastava et al.,
2015b). Among various advanced strategies for cellulase
production, solid-state fermentation (SSF) is a potential
and cost-effective approach (Pandey, 1994). The SSF is
done without the presence of free water in large amount
(Pandey et al., 2000) but in the presence of ample amount
of moisture which provides support for the growth of
fungi on lignocellulosic substrate (Singhania et al., 2009).
Because of this, the cost of dewatering step during the
downstream processing can be significantly
reduced.Although, at the commercial scale cellulase
production is carried out in the submerged fermentation,
but due to the low yield and high cost this process is not
economical. On the other hand, SSF has the efficiency to
be up-scaled for greater volume production. Additionally,
SSF is advantageous for higher enzymes concentration,
higher productivity as well as low requirement of the
sterility equipments (Holker et al., 2004). Besides, the
crude enzyme components obtained from the SSF can be
directly used for the hydrolysis of the lignocellulosic
substrate. Further, it is expected that the production cost
of SSF can be reduced by tenfold compared to the
submerged fermentation (Raghavarao et al., 2003) due to
the lower energy consumption (Holker et al., 2004) and
suitable low cost lignocellulosic substrate (Singhania et
al., 2010). SSF provides a suitable condition for
filamentous fungi due to easy cultivation (Holker et al.,
2004). Filamentous fungi such as T. reesei, A. niger, A.
fumigates are well-known fungi for cellulase production
under the SSF. Several studies have been reported on
effective cellulase production under the solid-state
fermentation (Ang et al., 2013; Srivastava et al., 2014;
Chandra et al., 2010). Besides SSF, enhancement of
cellulase production depends on selection of
lignocellulosic materials used for SSF. Lignocellulosic
biomass are found in huge quantity and considered as the
potential substrates for biofules industries. These
biomasses are obtained as waste products of agricultural
practices, especially fromvarious agriculture based
industries (Perez et al., 2002). Since these biomasses are
natural and renewable resources of energy, they are the
focal point of modern industries. Additionally, these
lignocellulosic biomasses can effectively be converted
into different value-added products such as cellulase
production, sugar generation for bio-fuels production as
well as cheap energy sources for microbial fermentation
and enzyme production (Asgher et al., 2013; Iqbal et al.,
2013). Thus, addition of suitable lignocellulosic substrate
under the SSF can improve the cellulase production in
greater extent. Apart from SSF and suitable substrate,
cellulase production can be further enhanced by using the
genetically modified organisms. Although, many
filamentous fungi are able to produce cellulase but due to
the lower yield, feasibility in the area of
commercialization is not possible. Insertion of high
cellulase producing gene into thermophilic organismcan
improve the cellulase production, but very few
information is available in the literatures. Therefore, a
good understanding about the genetics of the organismis
needed. Improvements in specific activities of cellulose
can be possible via cellulase engineering which depend
on rational design. The concept of artificial designing of
cellulase seems to be more promising for cellulases
having desired features. In addition with genetic
modification, co-culture concept is advantageous for

International Journal of Recent Trends in Science And Technology, P-ISSN 2277-2812 E-ISSN 2249-8109
Special Issue, ACAEE: 2018 pp 17-25
Copyright © 2018, Statperson Publishing Corporation, International Journal of Recent Trends in Science And Technology, P-ISSN 2277-2812 E-ISSN 2249-8109, Special Issue, ACAEE: 2018
cellulase production under the SSF. Improvement in
cellulase production can be achieved via co-culture of
different fungi in the single medium (Kalyani et al.,
2013). Co-culture also has several advantages, e.g. higher
productivity, adaptability and substrate utilization
compared to pure culture (Holker et al., 2004). The
enzymes system obtained via co-culturing of different
fungi may interact with each other and forms a complete
cellulase system. The performance of co-culturing fungi
for cellulase production has also been reported by (Hu et
al., 2011). These authors found that the lignocellulosic
components were depolymerized to a greater extent
whenthe fungi were co-cultured on lignocellulosic
substrate during the SSF and showed better efficiency. In
one of the other study by (Kalyani et al., 2013), β
glucosidase activity was recovered by co-culturing of
Sistotrema brinkmannii and Agaricus arvensis. In
addition of various approaches to improve the cellulase
production at the industrial scale, number of different co-
factors such as addition of metal ions can also enhance
the cellulase production, significantly (Srivastava et al.,
2014).Recently, concept of nanomaterials has aroused as
a new era in the revolution of renewable energy
production. Recently, (Dutta et al., 2014) reported
enhanced cellulase production in the presence of
hydroxyapatite nanoparticles. In this study, an improved
thermal stability of cellulose was achieved along with
reducing sugars at the hydrolysis temperature of 80°C,
when rice husk/rice straw was used as the substrates. In
one of the very recent study, by (Srivastava et al., 2015b)
an improved celluase production, thermal stability as well
as sugar productivity in the presence of Fe3O4/alginate
nanocomposite has been reported. In this study, a higher
yield of cellulases using A. fumigatus AA001 under the
SSF was achieved in the presence of Fe3O4/alginate
nanocomposite. Further, cellulase production and its
thermal stability were also improved in the presence of
Fe3O4 nanoparticle and Fe3O4/ alginate nanocomposite.
The results reported by the authors clearly exposed that
nanoparticles can play an important role to improve the
cellulase production as well as the entire bioconversion
process. In addition, an improved cellulase production
and thermal stability has also been reported in the
presence of nickel cobaltite (NiCo2O4) nanoparticle
under the SSF using the thermotolarent A. fumigatus NS
(Class: Eurotiomycetes) (Srivastava et al., 2014). Thus,
nano particles may be considered as one of the key factor
to enhance the cellulase production in the near future.
Apart from the aforementioned studies there are other
literatures which have reported an improvement of
cellulase production, thermal stability as well as its
hydrolysis efficiency in the presence of nanoparticles
(Ansari and Husain 2012; Shakeel and Qayyum
2012;Verma et al., 2013). Ansari and Husain (2012) have
discussed the immobilization of cellulase enzyme in the
presence of magnetic nanoparticles. These authors have
proposed that the nanoparticle works as a carrier and
improves not only the thermal stability but also the pH
stability and tolerance, frominhibitor during the
enzymatic hydrolysis. Verma et al. (2013) reported that
the thermal stability of - glucosidase enzyme was
increased in presence of magnetic nanoparticles and
exhibited half life of the same enzyme at 70°C. Although,
nanoparticles have shown potential for improved
cellulase production, thermal stability, and hydrolysis
efficiency, their mechanism is not well understood;
therefore, emphasize should be made on cellulase
production using the nanomaterials.
Genomic approaches: Genomic sequencing of
cellulolytic organisms has been carried during the past
decade and the genome sequences have provided
important new information about how microorganisms
degrade cellulose. The sequences of the aerobic
microorganisms: Hypocrea jecorina (Tricoderma reesei),
Phanerochaete chrysosporium, and Thermofida fusca, all
contain multiple cellulase genes, most of which encode a
carbohydrate binding module (CBM), and several
processive cellulase genes are present in each organism
(Martinez et al., 2004 and Likidis et al., 2007 ). The
genome sequences of Clostridium thermocellum,
Ruminococcus albus and Ruminococcus flavifaciens all
contain scaffoldin genes and multiple cellulases genes
that encode docerin domains, consistent with the presence
of cellulosomes in these anaerobic bacteria and several
processive cellulase genes are found among the docerin
encoding genes (Bayer et al., 2008). There are three
cellulolytic microorganisms whose genomes do not
contain known genes for processive cellulases or docerin
domains or scaffoldins: Cytophiga hutchinsonii is an
aerobic cellulolytic bacterium that is tightly bound to
cellulose fibers during growth on cellulose (Xie et al.,
2007), while Fibrobacter succinogenes is an anaerobic
cellulolytic bacterium that also is tightly bound to
cellulose fibers (Qi et al., 2007). From their genome
sequences these organisms do not use either the free
cellulase or the cellulosomal mechanism to degrade
cellulose, so they must use a novel mechanism (Wilson et
al., 2008). Finally Postia plancenta is an aerobic brown
rot fungus that appears to produce hydrogen peroxide and
Fe (II) ions that generate OH radicals that carry out
cellulose depolymerisation (Martinez et al. ,2009).
Further research is needed on each of these organisms to
determine the detailed mechanisms that they use to
completely metabolize cellulose. Metagenomics is also
being used to try to identify new cellulases and a major
study of DNA isolated from the microrganisms in termite

Ashiq Magrey, Sanjay Sahay, Ragini Gothalwal
International Journal of Recent Trends in Science And Technology, P-ISSN 2277-2812 E-ISSN 2249-8109, Special Issue, ACAEE: 2018 Page 22
guts was reported recently (Warnecke et al. , 2007).
About one hundred hydrolases related to cellulose
degradation were identified including members of eight
cellulose families; however, no members of families
containing exocellulase genes were present. This might
be due to the fact that termites chew up the biomass into
very fine particles that may be easier to degrade then
other forms of cellulose. It is interesting that screening of
genes for cellulase activity, either from isolated
organisms or from DNA libraries from various
environmental samples has not identified any new cellase
families in the past few years. One novel hydrolase
containing both a glucanase and a xylanase was found in
a library isolated from soil (Nam et al., 2009).
Non-cellulase protein stimulating cellulases: Several
proteins have been identified that appear to modify
cellulose and enhance its hydrolysis by cellulase. One is a
class of plant proteins called expansions (Carey et al. ,
2007 ). Another is a fungal protein with some homology
to expansin called swollenin (Yao et al. , 2008). Recently
an expansin like protein has been identified in Bacillus
subtilis and its structure was determined. In another
study, this protein was shown too stimulate corn stover
hydrolysis by crude cellulase (Kerff et al,.2008 and Kim
et al., 2009). Several organisms secrete proteins that only
contain CBMs and two T. fusca proteins (E7, E8) have
been purified and shown to stimulate low concentrations
of cellulases (Moser et al., 2008). Finally there are the
family 61 proteins mentioned earlier.
Modeling cellulase activity: Many attempts have been
made to model the cellulase catalyzed hydrolysis of
crystalline cellulose but we still do not know enough
about this process to create a true mechanistic model. Peri
et al. presented a detailed mechanistic model of
amorphous cellulose hydrolysis by crude cellulase that
fits their experimental results quite well (Peri et al. ,
2007).
Cellulase engineering: There are three main approaches
that are being used to engineer cellulases with higher
activity on crystalline cellulose: directed evolution,
rational design, and increasing cellulase thermostability
by either of the preceding methods, which can also lead to
higher activity. Engineering more thermostable enzymes
is relatively straightforward and there are some general
approaches that can be applied to any enzyme for which a
largenumber of related sequences are known, as is true
for most cellulases (Heinzelmanet al., 2009). A recent
paper describes evolving T. Reesei Cel12A for enhanced
thermostability while another evolved a family 5
endoglucanase with higher activity on CMC but it had no
activity on crystalline cellulose (Nakazawa et al. , 2009
and Lin et al., 2009). At this time there are no published
reports of engineered cellulases with major (greater than
1.5-fold) increases in activity on crystalline cellulose.
Furthermore, to be useful in an industrial process the
improved enzyme has to increase the activity of a
synergistic mixture containing several cellulases and in
several cases mutant enzymes with higher activity do not
do this (Zhang et al. , 2000). At this time, it is not clear
why this is happening but it has been shown for several
exocellulases. Another surprising result is that an
improved processive endocellulase catalytic domain,
produced by combining two site directed mutations, that
showed higher activity in synergistic mixtures then the
wild type catalytic domain, did not show higher activity
on crystalline cellulose then wild type intact enzyme
when the missing domains were added back to form the
intact mutant enzyme. This result seems surprising but it
shows that activity on crystalline cellulose may involve
interactions between the catalytic domain and the
carbohydrate binding module (CBM) that go beyond the
CBM simply anchoring the catalytic domain to the
cellulose (Esteghlalian et al. , 2001). Directed evolution
of cellulases with improved activity on crystalline
cellulose requires that the mutant cellulases be screened
on a crystalline substrate not on CMC as most mutations
that increase CMCase activity decrease activity on
crystalline cellulose. Furthermore, the native enzyme
should be utilized, not the catalytic domain given the
above result. Finally any improved enzymes need to
tested in the appropriate synergistic mixture on the actual
substrate for the final process in order to be certain that
they will be useful. A problem with directed evolution is
that it can only be used to screen potential single or with a
massive screen potential double mutations, since the
mutant library size required to include most possible
larger multiple mutations is too large. Rational design
does not have this limitation, but it does require a detail
understanding of structure–functional relationships for
cellulase crystalline cellulose activity that is still lacking.
If we can gain a clear understanding of exactly how
cellulases hydrolyze crystalline cellulose it should be
possible to design enzymes with multiple changes that
have higher activity on specific biomass substrates.
Designer cellulosomes: Another approach to engineering
more active cellulose degrading enzymes is to create
optimized cellulosomes by synthesizing hybrid scaffoldin
molecules that contain cohesins with different binding
specificity from different organisms. The exact
composition and geometry of the enzymes in a
cellulosome can be controlled by attaching the
approbriate docerin domain to each enzyme in the
cellulosome. In one experiment, the six T. fusca cellulases
produced during growth on cellulose were modified by
removing their family 2 CBM domain and replacing it
with a docerin domain, thus converting a free cellulase

International Journal of Recent Trends in Science And Technology, P-ISSN 2277-2812 E-ISSN 2249-8109
Special Issue, ACAEE: 2018 pp 17-25
Copyright © 2018, Statperson Publishing Corporation, International Journal of Recent Trends in Science And Technology, P-ISSN 2277-2812 E-ISSN 2249-8109, Special Issue, ACAEE: 2018
system into a cellulosomal system. There were a number
of interesting findings from this approach but it did not
produce a cellulosome with increased cellulase activity
over the free cellulase system (Caspl et al., 2008).
Another experiment involved adding CBM domains to
two of the key cellulosomal enzymes. This inceased the
activity of each enzyme when it was bound to an artificial
scaffoldin containing one cohesin but various designer
cellulosome containing the modified enzymes and other
cellulases all had lower activity on crystalline cellulase
than comparable designer cellulosomes containing the
WT enzymes (Mingardon et al., 2007 ). These results do
not invalidate the possibility of improving the activity of
cellulosomes by the designer approach but we need to
understand more about how the enzymes on cellulosomes
interact to degrade crystalline cellulose before we can
create better cellulosomes.
CONCLUDING REMARKS
This review focused on the application of cellulase in
biofuels production and its industries. Specifically, the
demand of cellulase is gradually increasing in biofuel
industry and therefore, development of cost-effective
methods to produce cellulase at large scale is needed.
Although, it is challenging to develop the cost-effective
technology and economy in biofuels industries,
continuous efforts are being made in this field. The use of
cheaper and waste material, thermotolraent/thermophilic
organisms, thermostable/thermophilic enzyme, and
addition of certain co-factor can enhance the cellulase and
consequently the biofuels production. In addition, the
genetic modification of cellulase producing microbes is
another potential area to explore the higher amount of
cellulase for biofuels production.
ACKNOWLEDGEMENT
The authors are thankful to the department of
Biotechnology Barkatullah university Bhopal (M.P) and
department of Botany Government NSCB P.G college
Biora, Rajgarh, (M.P) whose comment and suggestion
make this study more valuable.
REFERENCES
1. Ang T.N., Ngoh G.C. and Chua A.S.M. (2013).
Development of a novel in oculum preparation method
for solid-state fermentation. Cellophane film culture
(CFC) technique. Ind. Crop Prod., 43:774–777.
2. Asgher M.,Ahmad Z. and Iqbal H.M.N. (2013). Alkali
and enzymatic delignification of sugarcane bagasse to
expose cellulose polymers for saccharification and bio-
ethanol production. Ind. Crop Prod.,44:488–495.
3. Ansari S.A. and Husain Q. (2012). Potential applications
of enzymes immobilized on/in nano materials: a review.
Biotechnology Adv. 30:512–523. doi: 10.1016/j.biotech
adv.2011.09.005.
4. Bayer EA, Lamed R, White BA, Flint HJ. (2008). From
cellulosomes to cellulosomics. Chem Rec 2008, 8:364-
377.
5. Begum F, Absar N, Alam MS. (2009). Purification and
characterization of extracellular cellulase from A. oryzae
ITCC-4857.01. JASR 5: 1645-1651.
6. Bayer, E.A., Belaich, J.P., Shoham, Y., and Lamed, R.
(2004). The cellulosomes multienzyme machines for
degradation of plant cell wall polysaccharide. Annual
Review of Microbiology 58: 521–554.
7. Bayer, E.A., Morag, F., and Lamed, R. (1994). The
cellulosome—a treasure-trove for biotechnology. Trends
in Biotechnology 12 (9): 379–386.
8. Chen Yao, Stipanovic Arthur J, Winter William T,
Wilson David B, Kim Young-Jun. (2007). Effect of
digestion by pure cellulases on crystallinity and average
chain length for bacterial and microcrystalline celluloses.
Cellulose 2007, 14:283-293.
9. Carvalho, L.M.J., Deliza, R., Silva, C.A.B., Miranda, M,
and Maia, M.C.A. (2003). Identifying the adequate
process conditions by consumers for pineapple juice
using membrane technology. Journal of Food Technology
1:150–156.
10. Carey RE, Cosgrove DJ (2007). Portrait of the expansin
superfamily in Physcomitrella patens comparisons with
angiosperm expansins. Ann Bot (Lond), 99:1131-1141.
11. Caspi J, Irwin D, Lamed R, Li Y, Fierobe HP, Wilson
DB, Bayer EA (2008). Conversion of Thermobifida fusca
free exoglucanases into cellulosomal components
comparative impact on cellulose-degrading activity.
Journal of Biotechnolgy 135:351-357.
12. Chandra M., Kalra A., Sharma, P.K., Kumar H. and
Sangwan, R.S. (2010). Optimization of cellulases
production by Trichoderma citrinoviride on marc of
Artemisia annua and its application for bioconversion
process. Biomass Bioenerg., 34:805–811.
13. Dutta N., Mukhopadhyay A., Dasgupta A.K. and
Chakrabarti K. (2014). Improved production of reducing
sugars from rice husk and rice straw using bacterial
cellulase and xylanase activated with hydroxyapatite
nano particles. Bioresourc. Technol., 153:269–277
14. Esteghlalian AR, Srivastava V, Gilkes NR, Kilburn DG,
Warren RA, Saddle JN (2001). Do cellulose binding
domains increase substrate accessibility? Appl Biochem
Biotechnol, 91 93:575-592.
15. Elder, Chahal, D.S., and Ishaque, M. (1986). Integrated
processes for production of edible protein and fuel
ethanol from biomass. Eutropic 22: 130-131.
16. Henrissat, B., Teeri, T.T., and Warren, R.A.J. (1998). A
scheme for designating enzymes that hydrolyse the
polysaccharides in the cell walls of plants. FEBS Letters
425 (2): 352–354.
17. Heinzelman P, Snow CD, Wu I, Nguyen C, Villalobos A,
Govindarajan S, Minshull J, Arnold FH (2009). A family
of thermostable fungal cellulases created by structure-
guided recombination. Proc Natl Acad Sci U S A,
106:5610-5615.
18. Holker U., Hofer M. and Lenz J. (2004).
Biotechnological advantages of laboratory-scale solid-

Ashiq Magrey, Sanjay Sahay, Ragini Gothalwal
International Journal of Recent Trends in Science And Technology, P-ISSN 2277-2812 E-ISSN 2249-8109, Special Issue, ACAEE: 2018 Page 24
state fermentation with fungi. Appl. Microbiol.
Biotechnol., 64(2):175–186.
19. Hu H.L., Brink J.V.D., Gruben B.S.,Wosten H.A.B., Gu
J.D. and Vries R.P.D. (2011). Improved enzyme
production by co-cultivation of Aspergillus niger and
Aspergillus oryzae and with other fungi. Int. Biodeterior.
Biodegrad., 65 (1):248–252.
20. Iqbal H.M.N., Kyazze G. and Keshavarz T. (2013).
Advances in valorization of lignocellulosic materials by
bio-technology: An overview. Bio. Resources.,
8(2):3157–3176.
21. Jeoh T, Wilson DB, Walker LP (2006) Effect of cellulase
mole fraction and cellulose recalcitrance on synergism in
cellulose hydrolysis and binding. Biotechnol Prog
22:270-277.
22. Joshi V, Pandey A (1999) Biotechnology: Food
Fermentation:Microbiology, Biochemistry, and
Technology. Educational Publishers and Distributors.
23. Klyosov, A.A. (1995) Principles of the Enzymatic
Degradation of Cellulose. In Industrial Enzyme
engineering.
24. Kubicek, C.P. (1993) From cellulose to cellulase
inducers: facts and fiction. In Proceedings of the 2nd
Symposium Trichoderma reesei Cellulases and Other
Hydrolases (TRICEL ’93),
25. Kuhad, R.C., Manchanda. M., and Singh. A. (1999)
Hydrolytic potential of extracellular enzymes from a
mutant strain of Fusarium oxysporum. Bioprocess
Engineering 20 (2) 133 135
26. Kalyani D., Lee K.M., Kim T.S., Li J., Dhiman S.S.,
KangY.C. and Lee J.K. (2013)Microbial consortia for
saccharification of woody biomass and ethanol
fermentation. Fuel., 107:815–822.
27. Kim ES, Lee HJ, Bang WG, Choi IG, Kim KH (2009)
Functional characterization of a bacterial expansin from
Bacillus subtilis for enhanced enzymatic hydrolysis of
cellulose. Biotechnol Bioeng, 102:1342-1353.
28. Kerff F, Amoroso A, Herman R, Sauvage E, Petrella S,
File´e P, Charlier P, Joris B, Tabuchi A, Nikolaidis N,
Cosgrove DJ (2008) Crystal structure and activity of
Bacillus subtilis YoaJ (EXLX1), a bacterial expansin that
promotes root colonization. Proc Natl Acad Sci U S A,
105:16876-16881.
29. Kuhad RC., Gupta R., Singh A., (2011). Microbial
cellulases and their industrial applications. Enzyme
Research.
30. Lynd, L.R., and Zhang, Y. (2002) Quantitative
determination of cellulase concentration as distinct from
cell concentration in studies of microbial cellulose
utilization: analytical framework and methodological
approach. Biotechnology and Bioengeneering 77: 467-
475.
31. Lykidis A, Mavromatis K, Ivanova N, Anderson I, Land
M, Di Bartolo G, Martinez M, Lapidus A, Lucas S,
Copeland A et al (2007) Genome sequence and analysis
of the soil cellulolytic actinomycete Thermobifida fusca
YX. J Bacteriol, 189:2477-2486.
32. Lin L, Meng X, Liu P, Hong Y, Wu G, Huang X, Li C,
Dong J, Xiao L, Liu Z (2009) Improved catalytic
efficiency of endo-beta-1,4- glucanase from Bacillus
subtilis BME-15 by directed evolution. Appl Microbiol
Biotechnol, 82:671-679.
33. Moser F, Irwin D, Chen S, Wilson DB (2008) Regulation
and characterization of Thermobifida fusca carbohydrate-
binding module prot eins E7 and E8. Biotechnol Bioeng,
100:1066-1077.
34. Mai, C., Kues, U., and Militz, H. (2004) Biotechnology
in the wood industry. Applied Microbiology and
Biotechnology 63: 477-494.
35. Martinez D, Larrondo LF, Putnam N, Gelpke MD, Huang
K, Chapman J, Helfenbein KG, Ramaiya P, Detter JC,
Larimer F et al (2004) Genome sequence of the
lignocellulose degrading fungus Phanerochaete
chrysosporium strain RP78. Nat Biotechnol, 22:679-680.
36. Moser F, Irwin D, Chen S, Wilson DB (2008) Regulation
and characterization of Thermobifida fusca carbohydrate-
binding module proteins E7 and E8. Biotechnol Bioeng,
100:1066-1077.
37. Mingardon F, Chanal A, Tardif C, Bayer EA, Fierobe HP
(2007) Exploration of new geometries in cellulosome-
like chimeras. Appl Environ Microbiol, 73:7138-7149.
38. Nieves R.A., Ehrman C.I.,AdneyW.S., Elander R.T. and
HimmelM.E. (2009) Technical communication: survey
and analysis of commercial cellulase preparations
suitable for biomass conversion to ethanol. World J.
Microb. Biot., 14:301–304.
39. Nam KH, Kim SJ, Hwang KY (2009)Crystal structure of
CelM2, a bifunctional glucanase-xylanase protein from a
metagenome library. Biochem Biophys Res Commun
2009, 383:183-186.
40. Pandey, A., Selvakumar, C.P., Soccol, R., and Nigam P.
(1999) Solid state fermentation for the production of
industrial enzymes. Current Science 77(1): 149-162.
41. Peri S, Karra S, Lee YY, Karim MN (2007) Modeling
intrinsic kinetics of enzymatic cellulose hydrolysis.
Biotechnol Prog 2007, 23:626-637.
42. PandeyA., Srivastava N. and Sinha, P. (2012)
Optimization of photo-fermentative hydrogen production
by Rhodobacter sphaeroides NMBL-01. Biomass
Bioenerg., 37:251–256.
43. Payne C.M., Knott BC,Mayes H.B., Hansson H.,
HimmelM.E., SandgrenM., Stahlberg J. and Beckham
G.T. (2015) Fungal Cellulases. Chem. Rev.,
115:1308"1448
44. PandeyA. (1994) Solid-state fermentation: An overview.
In: PandeyA (Ed.), Solid-State Fermentation. Wiley
Eastern Limited, New Delhi, p 3–10.
45. Pandey A., Soccol C.R. and Mitchell D. (2000) New
developments in solid-state fermentation: Ibioprocesses
and products. Process Biochem., 35(10):1153–1169.
46. Perez J.,Munoz-D.D.LR.T. andMartýnez J. (2002)
Biodegradation and biological treatments of cellulose,
hemicellulose and lignin: An overview. Int. Microbiol.,
5:53–63.
47. Qi M, Jun HS, Forsberg CW (2007) Characterization and
synergistic interactions of Fibrobacter succinogenes
glycoside hydrolases. Appl Environ Microbiol 2007,
73:6098-6105.
48. Raghavarao K., Ranganathan T.V. and Karanth N.G.
(2003) Some engineering aspects of solid-state
fermentation. Biochem. Eng. J., 13(2–3):127–135.
49. Sang-Mok, L., and Koo Y.M. (2001) Pilot-scale
production of cellulase using Trichoderma reesei Rut C-

International Journal of Recent Trends in Science And Technology, P-ISSN 2277-2812 E-ISSN 2249-8109
Special Issue, ACAEE: 2018 pp 17-25
Copyright © 2018, Statperson Publishing Corporation, International Journal of Recent Trends in Science And Technology, P-ISSN 2277-2812 E-ISSN 2249-8109, Special Issue, ACAEE: 2018
30 in fed-batch mode. Journal of Microbiology and
Biotechnology 11: 229–233.
50. Srivastava N., Rawat R., Sharma R., Oberoi H.S.,
Srivastava M. and Singh J. (2014) Effect of
nickelcobaltite nanoparticles on production and
thermostability of cellulases from newly isolated
thermotolarent Eurotiyomycetes sp.NS. Appl. Biochem.
Biotechnol., 174:1092–1103.
51. Sukumaran, R. K. and Sighania R. R., Pandey A. (2005)
Microbial cellulases - Production, applications and
challenge. Journal of Scientific and Industrial Research,
64(11), 832-844
52. Sannigrahi, P., Kim, H., Jung, S., and Ragauskas A. J.
(2011) Pseudo-lignin and pretreatment chemistry. Energy
Environment and Science 4:1306-1310.
53. Schubert, C. (2006) Can biofuels finally take center
stage? Nature Biotechnology 24: 777 784.
54. Srivastava N., Singh J., Srivastava M., Ramteke P.W. and
Mishra P.K. (2015b) Improved production of reducing
sugars fromrice strawusing crude cellulase activated with
Fe3O4/Alginate nanocomposite. Bioresour. Technol.,
183:262–266.
55. Srivastava N., Rawat R., Oberoi H.S. and Ramteke P.W.
(2015a) A review on fuel ethanol production from
lignocellulosic biomass. Int. J. Green En. 12:949–960.
56. Singhania R.R., Patel A.K., Soccol C.R., PandeyA.
(2009) Recent advances in solid-state fermentation.
Biochem. Eng. J., 44(1):13–18.
57. Singhania, R.R., Sukumarana, R.K., Patel A.K.,
Larrocheb C. and Pandey, A. (2010) Advancement and
comparative profiles in the production technologies using
solid-state and submerged fermentation for microbial
cellulases. Enzyme Microb. Tech., 46:541–549.
58. ShakeelA.A. and QayyumH. (2012) Potential
applications of enzymes immobilized on/in nano
materials: A review. Biotechnol. Adv., 30:512–523.
59. Srivastava N., Rawat R., Sharma R., Oberoi H.S.,
Srivastava M. and Singh J. (2014) Effect of
nickelcobaltite nanoparticles on production and
thermostability of cellulases from newly isolated
thermotolarent Eurotiyomycetes sp.NS. Appl. Biochem.
Biotechnol., 174:1092–110
60. VermaM.L., ChaudharyR., Tsuzuki T., BarrowC.J. and
PuriM. (2013) Immobilization of -glucosidase on a
magnetic nanoparticle improves thermostability:
Application in cellobiose hydrolysis.Bioresour. Technol.,
135:2–6.
61. Warnecke F, Luginbu¨ hl P, Ivanova N, Ghassemian M,
Richardson TH, Stege JT, Cayouette M, McHardy AC,
Djordjevic G, Aboushadi N et al., (2007) Metagenomic
and functional analysis of hindgut microbiota of a wood-
feeding higher termite. Nature, 450:560-565.
62. Wilson DB (2009): Cellulases. In Encyclopedia of
Microbiology, 3rd edn. Edited by Schaechter M. San
Diego: Elsever Inc; 2009.
63. Wilson DB (2008) Three microbial strategies for plant
cell wall degradation. Ann N Y Acad Sci, 1125:289-297.
64. Xie G, Bruce DC, Challacombe JF, Chertkov O, Detter
JC, Gilna P, Han CS, Lucas S, Misra M, Myers GL et al.,
(2007). Genome sequence of the cellulolytic gliding
bacterium Cytophaga hutchinsonii. Appl Environ
Microbiol, 73:3536-3546.
65. Yao Q, Sun TT, Liu WF, Chen GJ (2008). Gene cloning
and heterologous expression of a novel endoglucanase,
swollenin, from Trichoderma pseudokoningii S38. Biosci
Biotechnol Biochem, 72:2799-2805.
66. Zhang S, Irwin DC, Wilson DB (2000). Site-directed
mutation of noncatalytic residues of Thermobifida fusca
exocellulase Cel6B. Eur J Biochem, 267:3101-3115.
Source of Support: None Declared
Conflict of Interest: None Declared