INTRODUCTION

CELLULASES

ENDOGLUCANASE

Lignocellulosics are the most abundant biopolymers onEarth. Their hydrolysis into fermentable sugars, phenolicsand sugar acids is necessary for using them as substrates in theproduction of industrially important value added products.Cellulases, hemicellulases and ligninases are the group ofenzymes which carry out enzymatic hydrolysis oflignocelluloses in combination, and these enzymescollectively are known as 'lignocellulolytic enzymes'.Amonglignocellulolytic enzymes, cellulases are the third mostsignificant and key enzymes in the conversion of cellulose tosimple sugars (Chinedu 2005). Cellulases are the groupof enzymes which

internal regions of the fibrils,

h hydrolyse cellobiose and othercellodextrins with a low degree of polymerizations to glucose(Szijarto 2004).

Among the estimated 3.0 million species of fungi extant onEarth (Hawksworth, 2012), more than 100,000 have so farbeen described, and among them, approximately 50 speciesare capable of growth in the temperature range of 40-60 ºC(Mouchacca, 2000). Thermophilic fungal species depictrather broad cardinal (minimum, maximum and optimum)temperatures which extend from 20 ºC to upwards of 50 ºC(Cooney and Emerson, 1964). A thermophilic mould growswell at 50 ºC and beyond, but considered not to grow below 20ºC. The heat tolerant enzymes produced by thermophilicfungi are of immense interest in industrial bioprocesses.

Thermophilic fungi have evolved adaptations in response tovaried environmental conditions like extreme pH, high saltconcentrations and temperatures, which are hostile for most

of the organisms, but allow them to survive and colonizevarious natural substrates. The search for industriallyimportant enzymes such as lipases, cellulases, xylanases,amylases, and proteases led to the discovery of thermostableenzymes (Johri 1999; Singh 2016; Singh 2016).Thermostable enzymes have the advantages over these frommesophilic fungi because of their action at high temperaturesleads to decrease in the viscosity of media and acceleration inthe reaction rates, besides minimizing contamination risks(Fernandes 2008). Both biochemical and molecular(genomic) studies have confirmed the ability of these mouldsin efficiently degrading a variety of lignocellulosic substrates(Singh 2016).

(Moretti 2012) and(Van Den Brink 2013) have a high

potential in the bioconversion of lignocellulosic biomass intofermentable sugars. is anotherpromising fungus which is capable of producing thermostableenzymes (McClendon 2012). Furthermore, thegenomes of and havebeen analysed recently (Berka 2011), which showed theevidence for their ability to degrade all the majorpolysaccharides present in lignocellulosic biomass. Thesestudies and data reinforce and encourage the continued searchfor thermostable enzymes from thermophilic fungi. Thisreview focuses on the production, characteristics andpotential applications of cellulolytic enzymes of thermophilicmoulds.

Cellulases are a complex enzyme system that includes exo, end ,

(Maheshwari 2000). These are brieflydescribed below:

ly hydrolyses carboxymethyl cellulose (CMC) or

et al.,

et al.,

et al., et al.,

et al.,

et al.,Myceliophthora thermophila et al., M.heterothallica et al.,

Thermoascus aurantiacus

et al.,M. thermophila Thielavia terrestris

et al.,

et al.,

,

hydrolyze β-1,4- glycosidic bondsof intactcellulose and other cellooligosaccharides. For completedegradation of cellulose, three main enzymes [exoglucanase(EC 3.2.1.91), endoglucanase (EC 3.2.1.4), and β-glucosidase (EC 3.2.1.21)] act synergist ical ly.Endoglucanases favourably cleave β-glucosidic bonds andhydrolyse the exoglucanases(cellobiohydrolases) release cellobiose from terminal ends ofchains, while the process of degradation is completed by β-glucosidases whic

-1,4-β-D-glucanase o-1,4-β-D glucanase and β-D-glucosidase

Endoglucanase (endo-β-1, 4-D-glucanase or endo-β-1, 4-D-glucan-4- glucano-hydrolase) usually known as CMCaserandom

KAVAKA48(2): 47-58(2017)

Bijender Singh ,Anju Bala , Seema Dahiya and T. Satyanarayana

Production, characteristics and potential applications of the cellulolytic enzymes of thermophilicmoulds

1 1 1 2*

1

2Laboratory of Bioprocess Technology, Department of Microbiology, Maharishi Dayanand University, Rohtak-124001, IndiaDivision of Biological Sciences and Engineering, Netaji Subhas Institute of Technology, Azad Hind Fauz Marg, Sector 3,Dwarka, New Delhi-110078, India*Corresponding author Email.: tsnarayana@gmail.com(Submitted in February, 2017;Accepted on July 10, 2017)

ABSTRACTThermophilic moulds are capable of degrading plant organic residues at elevated temperatures by secreting a large array of hydrolases. Thesemoulds produce cellulolytic enzymes in submerged as well as solid substrate fermentations. Cellulases from these moulds have been characterized,which display a high thermostability, catalytic activity and fast rate of reactions as compared to their mesophilic counterparts. Their optimalactivities lie in the range of 50-70 C and pH of3.0-6.0 with molecular masses between 40 and 240 kDa.The genes encoding cellulolytic enzymes of themoulds have been cloned and expressed in both homologous and heterologous systems. Cellulolytic enzymes of thermophilic moulds have beenshownto be efficient in plantbiomass degradation.The applicability ofcellulases in a numberofbiotechnologicalprocesses such as the production ofbiofuels, food andanimal feeds,detergents and paperpulp processing has been demonstrated.

KEYWORDS:

o

Thermophilic moulds, cellulases, lignocellulosics, solid state fermentation, submerged fermentation, biofuels, bioethanol

47

swollen cellulose resulting in the increase of concentration ofreducing sugars by decreasing the length of polymer (Robsonand Chambliss, 1989; Begum 2009). Cellodextrins, theintermediate products of cellulose hydrolysis, are alsohydrolysed by this enzyme and converted into glucose andcellobiose (disaccharide), but it is not active on crystallinecelluloses such as cotton or avicel.

tes, but they do not cause hydrolysis ofsoluble derivatives of cellulose such as CMC andhydroxyethyl cellulose. As a minor component, somecellulase systems also contain glucohydrolase (exo-1, 4-D-glucan-4-glucohydrolase) as a minor component (Joshi andPandey, 1999).

( that cleavesglucose from the non-reducing end (i.e., with a free hydroxylgroup at C-4) of cellooligosaccharides and cellobiose. Alkyland

1993).

Submerged fermentationis the frequently used process for production of industriallyimportant enzymes on a large scale. Generally, this process iscarried out in the presence of liquid medium having solublesubstrates which flow freely. The factors which makes it moreattractive includes the facility of controlling parameters of theprocess, monitoring and downstream processing (Sukumaran

2005). The main drawback in this process is that itrequires prolonged time for fermentation or gestation periodwith less productivity of enzyme (Singhania 2010). Inorder to attain superior productivity in SmF, various naturaland synthetic carbon sources have been used.

was first reported as the most active cellulaseproducer by Tong and Cole (1982) among several fungitested. Its optimum temperature for growth was 45 °C, whilemaximum cellulase from filter paper w

5.0 and 4.3, respectively. Bajaj (2014) reportedLAR5 to efficiently utilize low-

cost agricultural residues (wheat bran, maize bran, and ricehusk) for cellulase production. Maximum production wasobserved in wheat bran (2000 U/l) followed by maize bran(1800 U/l) and rice husk (1600 U/l). The production ofenzyme was enhanced substantially by the addition ofpeptone, mustard cake and soybean meal in medium.Cellulase was optimally active at 60 -70 ºC and pH 5.0. Buskand Lange (2013) studied the cellulolytic potential of sixteen

thermophilic fungal strains on a medium containingmicrocrystalline cellulose as the only carbon source, andreported that showed thehighest optimal endoglucanase activity at pH 8.0. Matsakas

(2015) optimized growth conditions of forcellulase production in SmF. The optimum temperature, pHand agitation for cellulase activity were 65 ºC, pH 5.5 and200, respectively. Among different carbon sources used foroptimization, Brewer's spent grain supported highest enzymeproduction (0.11±0.01 FPU/ml) during fifth and sixth day ofcultivation. Bala and Singh (2016

glucosidase (BGL) using cane molassesmedium and reported 36,420, 32,420 and 5,180 U Lproduction of CMCase, FPase and BGL, respectively. Theoptimal activity of these enzymes was reported at pH 5.0 and60 ºC. The addition of Tween 80 further enhanced theproduction of the cellulolytic enzymes. Zambare (2011)reported cellulase production from thermophilic consortiumand maximum production (up to 367 U L on prairie cordgrass) was achieved at 60 ºC and pH 4.0.produced maximum cellulase at 45 ºC in presence of 1%solka/floc supplemented with urea after 2-4 days (Coutts andSmith, 1976). While Kawamori . (1987) reported that

A-131when cultivated with 4 % alkali treatedbagasse at 45°C in 4 days produced about 70 U ml ofCMCase and it had excellent thermostability as there was noloss of activity after exposure to 60 °C for 24 h. Grigorevski-Lima (2009) reported the production of a thermostableand acid endoglucanase by isolatedfrom sugar cane bagasse. Highest levels of endoglucanase(365 U L ) was attained within 6 days of cultivation at 1 %sugarcane bagasse and 1.2 % corn steep liquor at pH 2.0 and65 °C.

For the enhancedproduction of various enzymes, one of the importantstrategies employed in the industries is SSF. This process isperformed in the absence of free water on the solid substrateswhich acts as solid support as well as source of nutrients forthe growth of microorganisms. SSF is gaining more attentionin the recent years, as it is an acceptable strategy for the reuseof the nutrient-rich agricultural residues with less energyinput and facilitates bioconversion of agricultural residues tovalue-added products (Pandey, 2003). Because of lowoperating cost and capital investment, SSF is an attractivestrategy to produce cellulases economically (Xia and Cen,1999). The physico-chemical properties of substrates such ascrystallinity, bed porosity and large surface area can affect theyield of cellulolytic enzymes by the moulds. In SSF, thegrowth and production of enzymes are influenced by theculture conditions such as pH, temperature, water activity andmoisture content which are crucial factors. The majorchallenges in SSF are the generation of heat and presence ofoxygen in the open space between substrate particles i.e.,porosity and these challenges have to be addressed properly(Thibault 2000; Oostra 2001). The mostcommonly used agricultural residues used as substrates forcellulase production are brans and straws (wheat and rice),corn stover, sawdust, and sugarcane baggase. Moretti

et al.,

et al.,

et al.,

et al.,

Thermoascusaurantiacus

et al.Sporotrichum thermophile

Thermomucor indicae-seudaticaeet

al. M. thermophila

et al.

M. thermophila

et al T.aurantiacus

et al.Aspergillus fumigatus

et al., et al.,

et al.

EXOGLUCANASE

GLUCOSIDASE

PRODUCTION OFCELLULASES BYTHERMOPHILICMOULDS

Submerged fermentation (SmF):

Solid State Fermentation (SSF):

Exoglucanase (exo-β-1, 4-D glucanase or cellobiohydrolase)acts from the non-reducing end of chain of cellulose splitting-off the cellobiose units. Exoglucanases are also active onswollen cellulose and cellodextrins and partially degradeamorphous substra

The complete hydrolysis of cellulose is accomplished by β-glucosidase β-D-glucoside glucohydrolase)

aryl β-glucosides are also hydrolysed by these enzymes(Kubicek

as produced at 40 °C.For β-glucosidase and carboxymethylcellulase (CMCase)activity, optimum temperature was 70 °C but it was 65 °C forfilter paper degrading activity (FPase). Maximum activity ofβ-glucosidase, and FPase and for CMCase was observed atpH

reported the production ofendo-β-1,4-glucanase (CMCase), exo-β-1,4-glucanase(FPase) and β-

β-

)

-1

-1

-1

-1

Production, characteristics and potential applications of the cellulolytic enzymes of thermophilic moulds48

(2012) isolated thermotolerant and thermophilic fungalstrains from soil, sugarcane piles and decaying organic matteron a medium having cardboard and corn straw as carbonsource at 45 °C. M.7.7 and

M.7.1 were identified as highestcellulase producers under SSF, and maximum enzymeactivity was recorded at pH 5.0 and 60 ºC and 70 ºC,respectively. The endoglucanases from these fungi werestable at 40 to 65 °C and pH 4.0 to 9.0.

M.7.7 produced54 U/g of CMCase in a medium that contained wheat bran andsugarcane bagasse. Kilikian (2014) reported the highestFPase (10.6 U/gdm) production by M77using sugarcane bagasse and soybean bran. This activity was4.4 times higher than that attained with wheat bran. Kaur(2015) studied the production of cellulases from

MTCC 1409 using rice straw as substrate andattained maximum production of cellobiase (9.30 U/g),CMCase (3.83 U/g) and FPase (1.6.7 U/g) at pH 6.0 and 45ºC. yielded high titres ofcellulase (42 U/g DMB) using citrus pectin and wheat bran in1:1 ratio after 4 days in SSF at pH 7.0 and 45 ºC (Kaur andSatyanarayana, 2004). Similarly, Pereira (2015)reported that JCP 1-4 was the highestproducer of endoglucanase (357.51 U g ),

). The highest endoglucanase (227.97 U g) and FPase (0.34 FPU) production

was recorded in 240, 192 and 96 h, respectively.P40M2 was

stable at pH 3.0 - 5.5 and at 40-60 °C (Delabona 2013).Reetika (2013) reported highest cellulolytic activity byDIA-4 strain at 45 °C in 72 h using a combination of wheatbran and sweet sorghum as substrate.

Thermophilic fungi secrete cellulases and hemicellulasesefficiently which are active under harsh conditions. Tanseyand Brock (1972) found that thermophilic moulds

, andhydrolyzed cellulose two times

faster than that of . Mandels (1975)reported thermophilic mould to hydrolyse polysaccharidefast, despite low cellulase titres in the culture filtrate.Purification and characterization of enzymes are consideredas important steps. The catalysts produced on nativelignocellulosic substrates are usually a mixture of variedcompounds together with proteins. Purification of asignificant macromolecule from such culture filtrates needsmultistep treatments by the classical ways includingprecipitat ion, quali ta tive analysis and columnchromatography (Maheshwari ., 2000). Moloney(1985) used Sephadex matrix for purification of proteins ofdifferent sizes by gel filtration chromatography. Thetechniques which are used for purification are totallydependent on the purification fold and yields of proteins.Cellulolytic enzymes from thermophilic moulds have been

purified using salt/solvent precipitation followed by ionexchange and gel filtration ( ).

Recombinant cellobiohydrolase II fromwas purified using ammonium sulphate

fract ionation and DEAE-Sepharose quick flowchromatography (Liu ., 2005). Cellobiosedehydrogenases from thermophilic mouldwere purified, and the genes encoding cellobiosedehydrogenases were successfully cloned and characterisedby Subramaniam . (1999). The purified enzyme showedoptimal activity at 60˚C. The characterization of enzymesshowed that molecular masses of enzymes were in the rangeof 91- 192 kDa and they were glycosylated. The activationenergy was 26.3 kJ/mol along with acidic isoelectric points of4.1 and 3.45 for enzymes I and II, respectively.

Cellulase is produced by in wheat bran as acarbon source.After processing with the combinations of ion-exhange and gel filtration chromatography,

wereobtained in good amounts from the culture extract (Hayashida

., 1988). produced multiple forms

, which were purified by ion exchangechromatography and separated multiple forms ofendoglucanases, which differed in carbohydrate content (28-51%), but had similar molecular masses (35 kDa by SDS-PAGE) with the pH optima between 5.5 - 5.8, temperatureoptima at 75-80˚C and iso-electric points (pI values) between2.8 and 3.2 (Moloney ., 1985). Endoglucanases werethought to be due to post- translational or post secretionalterations of one cistron product.

The combinations of various chromatographic techniqueswere used for purifications of glucosidase from

sp. using DEAE-Sepharose and PBE 94columns (Kaur ., 2007). The specific activity of purifiedenzyme was mg protein with 15.89 %yield. produced seven endoglucanasescloned from (Davies ., 1992;Schou ., 1998). All the endoglucanases were purified byaffinity chromatography. Similarly, Eriksen and Goksoyr(1976, 1977) purified different types of cellulases. Allcellulases in combination were able to degrade cottoncompletely as compared to those when used separately. Acellulase from RBB-1 was purifiedby ammonium sulphate precipitation, activity and sizeexclusion chromatography (Dave ., 2015). The recoveryand purification fold were 13.3 % and 6.6, respectively with amolecular mass of 35 kDa. Optimum temperature for activitywas 70˚C and stability was upto 80 ˚C for one h. Besideshigher stability at 80˚C, the protein also showed half-lifevalues of 192 and 144 h at 50 and 70 ˚C, respectively. Purifiedcellulase was optimally active at pH 4.0 with K and Vvalues of 37 mg/ml and 82.6 U/min/mg, respectively withhigher salt tolerance (Dave ., 2015). A consortium

using gel filtration chromatography (Bio-gel P-60 matrix) followed by SDS-PAGE with molecular masses of87 kDa for glucosidase, 78 kDa for cellulase I, 49 kDa for

Myceliophthora thermophilaAspergillus fumigatus

Aspergillus fumigatus

M. thermophila

et al.M. thermophila

et al.Humicola

fuscoatra

Myceliophthora thermophila

et al.M. thermophila

Aspergillus fumigatuset al.,

et al.

Sporotrichum thermophile Thermoascus aurantiacusChaetomium thermophile

Trichoderma viride

et al et al.

Chaetomiumthermophilum

et alM. thermophila

et al

Humicola insolens

et al Talaromyces emersonii

et al

Melanocarpuset al

Aspergillus oryzaeChateominm thermophile et al

et al

Thermoascus aurantiacus

et al

et alT.

aurantiacus

M.7.1 produced maximum β-glucosidase (40.4 U/g) andCMCase (40 to 47 U/g) when cultivated on the mixture ofwheat bran and corn straw.

β-glucosidase(45.42 U g ), β-glucosidase (34.36 U g

β-Glucosidase produced by

pureendoglucanase, exoglucanase, and β-glucosidase

of cellulases (endoglucanase, exoglucanases, and β-glucosidases)

β-

10.04 μmol min

ofcellulases including β-glucosidase were purified form

-1

-1 -1

-1

-1 -1PURIFICATION AND CHARACTERIZATION OFCELLULASES OFTHERMOPHILIC MOULDS

Table 1

-T

m max

Bijender Singh, Anju Bala, Seema Dahiya and T. Satyanarayana 49

Table 1 Purification of cellulolytic enzymes from thermophilic mouldsFungi Purification methods Enzyme Optimum

pHOptimum

temperature(˚C)

MW(kDa)

Enzymestability

References

Thermoascusaurantiacus

Cation exchangechromatography, gelfilteration

β-glucosidase 3-9 70 85 At 4°C forseveral weeks

Tong et al., 1980

Chaetomiumthermophilum CT2

Ammonium sulfate fraction,DEAE-Sepharose Fast flowchromatography

Recombinantcellobiohydrolase II

4 50 67 NR Liu et al., 2005

Myceliophthorathermophila ATCC48104

(NH4)2SO4 precipitation andcolumn chromatography onDEAE-Sephadex A-50,Sephadex G-200 and DEAE-Sephadex A-50

β-glucosidase 4.8 60 120 Purified enzymein the absence ofsubstrate wasstable up to 60°C

Roy et al., 1991

M. thermophilaD-14

Ammonium sulphateprecipitation , ion-exchangechromatography

Endoglucanase 4.8 65 100 Stable for 60 minat temperatures upto 70 ˚C

Roy et al., 1990

Melanocarpus sp.MTCC 3922

Ammonium sulphateprecipitation , ion-exchangechromatography

Endoglucanases(EG I and EG II)

6 and 5 50 and 70 40 and 50kDa

NR Kaur et al., 2007

Chaetomiumthermophile var.dissitum

Ion-exchangechromatography, gel filtration

Endo- andexoglucanases

NR NR 67 and 41 NR Erisken andGoksoyr, 1977

Humicola insolensYH-8

Avicel adsorption, heattreatments and ammoniumsulfate fractionation, Ionexchange chromatography

Avicelase, CMCase 5.3 foravicelase

and 5.0 forCMCase

50 NR Avicelase wasstable afterheating at 65°Cfor 5 min andCMCase retained45~ of the originalactivity afterheating at 95°Cfor 5 min

Hayasida andYoshioka, 1980

SporotrichumthermophileS.

S.

thermophile

thermophile

80% Ammonium sulphateprecipitation , ion-exchangechromatography

β-glucosidase 5.4 65 240 at 50 "C for up to6 h, but at 60"C, 25 YOof the The effectof pH onenzyme activitywas studied usingoriginal enzymeactivity was lostafter 2 h Stable at50 °C for 6h, 25%activity lost at 60°C after 2h

Bhat et al., 1993

Thermoascusaurantiacus RBB-1

ammonium sulfateprecipitation, ion exchangeand size exclusionchromatography

Endo-cellulase 4 70 35 Stability was up to80 °C for 1 h

Dave et al., 2015

Allesheria terrestris Ion exchangechromatography

Endo- andexoglucanases

NR NR 69 Inactivated onlyby 20% at 65°Cfor 6 h in theabsence of thesubstrate

Kvesitadze et al.,1997

Thermomyceslanuginosus-SSBP

ion exchangechromatography

β-D-glucosidase 6 65 200 30 min incubationat 50 ° C

Lin et al., 1999

cellulase II and 34 kDa for cellulase III. The enzymescontained varied carbohydrate contents of 33.0 to 5.5, 2.6 and1.8 %, respectively. Cellulases exhibited the ability todegrade paper and yeast glucan in combination but solelycellulases I and III were active on CMC. The Km values ofenzymes varied widely (Tong ., 1980).

sp.arrow pH range with optimal activity at pH 5.0 and 6.0

(Bhat ., 1993). It was stable for 240 min at 50 °C. Kaur., (2007) used isoelectric focussing gel technique for the

purification of of sp. Purifiedproduct showed one form of enzyme with acidic pI value.Usually cellulases produced by thermophilic moulds weremonomeric except glucosidase, which is dimeric(Maheshwari ., 2000).

ing ofsix subunits which dissociated to monomeric forms at highertemperatures (Kvesitadze ., 1997).

The endoglucanases with high molecular masses (30 to 100kDa) from thermophilic moulds were more thermostable andmostly active at 55-80°C and pH 5.0-5.5 and high

carbohydrate content upto 50 % (Li ., 2011).Exoglucanase fromthermophilic mouldswere able to toleratehigher temperature thanendoglucanase as theywere mostly active inbroad temperat urerange of 50 - 75 ºC witha molecular mass of 40 -7 5 k D a . A l lexog lucanases areconjugated proteins.The pH and temperatureoptima for all purec e l l u l a s e s f r o mthermophilic mouldswere similar ( ).The optimum pH forcellulases was in therange of 4.0-7.0 and 50-80 °C. The cellulasesfrom t hermophi l i cm o u l d s w e r ee x c e p t i o n a l l ythermostable and had alonger half life values athigher temperaturest h a n t h o s e o fmesophilic fungi.G l uc o s i da s e f ro m

f r o m

MTCC 3922 at pH 5.0and 60 ˚C (Lin ., 1999; Kaur ., 2007). The optimumpH and temperature were 5.0-6.0 and 50 ºC for

of the fungi (Bhat ., 1993).

Cellulase of was stronglyinhibited by Hg , but not affected by Zn . Similarly, Znalong with other metal ions such as sodium, potassium andcalcium did not affect -glucosidases of sp.MTCC 3922, but the activity was repressed by CuSO (Kaur

., 2007). Kaur . (2007) purified two endoglucanasesfrom sp. MTCC 3922. The molecular massesof two glucanases and their pI were ~40 and 50 kDa, and ~4.0and 3.6, respectively. Mercaptoethanol and SDS (denaturingagents) repressed one of the two endoglucnases (EG I) of

sp. MTCC 3922, the other (EG II) was notaffected. Affinity for degrading avicel by EG I and EG IIrevealed the existence of polysaccharide binding domains(CBD), although EG II lacked CBD (Kaur ., 2007). Roy

. (1990) purified endoglucanase from D-14 using ammonium sulphate precipitation followed by ionchromatography using DEAE-Sephadex. The pure enzymewas optimally active at pH 4.8 and 65˚C. Metal ions such as

et al

Melanocarpus

et al etal

Melanocarpus

et al Allescheria terrestris

et al

et al

T h e r m o m y c e slanuginosus

Me l anoc ar pus sp .

et al et al

et al

Paecilomyces thermophila

Melanocarpus

et al et alMelanocarpus

Melanocarpus

et alet al M. thermophila

produced β-glucosidase which was activein n

β-glucosidase

β-produced a

high molecular (210-230 kDa) β-glucosidase consis

was lessa c t i v e t h a n β -g l u c o s i d a s e

β-glucosidases of most

β

t

-t

Table 1

2+ 2+ 2+

4

Production, characteristics and potential applications of the cellulolytic enzymes of thermophilic moulds50

Hg , Fe , and Cu positively affected the enzyme activity.

Extracell was purified fromATCC 48104 using ammonium sulphate

precipitation and chromatographic techniques (Roy .,1991). The molecular mass was 120 kDa. Aldohexose acts asa competitive inhibitor. Enzyme activity was repressed byHgCl , FeSO and EDTA. Recombinant lucanase from

Y1 was purified by SP- Sepharose ionexchange chromatography (Xu ., 2015). The molecularmass was 33.5 kDa. Enzyme was thermostable at 60˚C for anh. Metal ions tested had no significant effect on the enzymeactivity except CTAB and Cu .

For the commercial applications of enzymes, researchersused hyper-producing microorganisms. Cellulases ofthermophilic moulds exhibited several desirable propertieswhich make their applicability in the saccharification oflignocellulosic biomass for the production of biofuels andother high value-added products. However, in some cases, theyields of enzymes are low, therefore, over-expressing in asuitable host is the ideal solution to overcome this problem.Consequently thermophilic moulds have received attention inisolating and cloning cellulase encoding genes from severalthermophilic fungi (Poças-Fonseca ., 2000; Berquist

2004). Recombinant DNA techniques provide the meansfor producing enzymesfrom thermophiles inmesophi les. Recentadvances in molecularb i o l o g y a n db i o t e c h n o l o g yfacilitated gene cloningand over expression.The cellulase genes ofthermophilic mouldswere expre ssed invarious mesophilic hostssuch as bacteria like

, yeasts likeand filamentous

f u n g i ( ) .Microbial cellulaseshave been classified as1, 3, 5, 6, 7, 8, 9, 10, 12,16, 44, 45, 48, 51, and 61types (Maheshwari

., 2000) Cellulasesf ro m t he rmo ph i l i cmoulds were mainlyclassified into 1, 3, 5, 6,7, 12, and 45 types(Maheshwari .,2000) M es oph i l i cfungus wasused as a host for theexpression of cellulaseg e n e s f r o m

(Haakana ., 2004) that resultedin improved cellulase activity as compared to parental strains.Cellulase encoding gene from was cloned,sequenced and finally expressed in(Hakkana ., 2004). Among two cellulase enzymes,Cel45A and Cel7A had molecular masses of 20 and 50 kDa,respectively, and the enzymes belong to different GH families45 and 7 (Hakkana ., 2004).

was used for the expression ofcellobiohydrolase from (Li .,2009). The pure recombinant enzyme displayed optimallyactive at pH 5.0 and 60˚C and it was active at highertemperature. Hong . (2007) used as acloning and expression host for endoglucanase of

. Recombinant endoglucanase waspurified by affinity technique with molecular mass of 35 kDathat belongs to GH5. Voutilainen . (2010) expressed the

Cel7A cellobiohydrolase in and thisexpression system allowed the structure guided proteinengineering by the introduction of further disulfide bridgeswithin the catalytic domain of the enzyme. Mutant proteinsconsisted of more disulphide bridges at totally differentpositions. Mutant enzymes displayed improved T (from 78.5to 84 ˚C) with higher half-lives at 70 ˚C than the wild. Themutants conjointly showed higher enzyme activity on Avicelat 75 ˚C. The cellulase gene from was purified andcloned in . Structural study of the enzyme showed

2+ 2+ 2+

2+

ular β-glucosidase

β-1,4-g

M.thermophila

et al

Humicola insolenset al

et al etal.,

E.coli Pichiapastoris

etal

et al.

T. reesei

Melanocarpus albomyces et al

M. albomycesTrichoderma reesei

et al

et alPichia pastoris

Chaetomium thermophilum et al

et al Kluveromyces

Thermoascus aurantiacus

et al T.emersonii S. cerevisiae

H. insolensA. oryzae

2 4

m

CLONING AND EXPRESSION OF CELLULASES OFTHERMOPHILIC MOULDS

Ta b l e 2

.

Fungi Gene Family Expressionvector

pI Thermal stability OptimumpH

Optimumtemperature

(˚ C)

MW(KDa)

References

Melanocarpusalbomyces

cel7b 7 Trichodermareesei

4.23 NR 6-8 NR 50.0 Haakana et al., 2004

M. albomyces cel45a 45 T. reesei 5.22 NR 6-8 NR 25.0 Haakana et al., 2004M. albomyces celL7b 7 T. reesei 4.15 NR 6-8 NR 44.8 Haakana et al., 2004Chaetomiumthermophilum

cel7a 7 T. ressei NR NR 4 65 54.6 Voutilainen et al., 2008

Talaromycesemersonii

cel7 7 E.coli 4.0 T1/2 : 68 min at80°C

5 68 48.7 Grassick et al., 2004

T. emersonii cel3a 3 T. reesei NR T1/2 : 62 min at65 °C

NR 71.5 90.5 Murray et al., 2004

T. emersonii cbh2 6A T. emersonii NR NR NR NR 47.0 Murray et al., 2003T. emersonii cel7A 7 Saccharomyces.

cerevisiaeNR T1/2: 30 min at

70°C4-5 65 46.8 Voutilainen et al., 2010

ChaetomiumthermophilumCT2

cbh2 NR Pichia pastorisGS115

NR 50% of its originalactivity after 30min at 70°C

4 50 67.0 Liu et al., 2005

C.thermophilum cbh3 NR P. pastoris 5.2 T1/2: 10 min at80°C

5 60 48.0 Li et al., 2009

Thermoascusaurantiacus

cbh1 7 S. cerevisiae 4.37 80% residualactivity for 60 minat 65°C

6 65 48.7.0 Hong et al., 2003a

T. aurantiacus eg1 5 S. cerevisiae 4.3 Stable for 60 min at70°C

6 70 37.0 Hong et al., 2003b

T. aurantiacusIFO9748

eg1/bgl1

3 P. pastoris NR 70% activity after 1h of incubation at60°C

5 70 NR Hong et al., 2007

T. aurantiacus bgl1 3 P. pastoris 4.61 70% residualactivity for 60 minat 60°C

5 70 93.5 Hong et al., 2007

T. aurantiacus cel7a 7 T. reesei 4.44 NR 5 65 46.9 Voutilainen et al., 2008T. aurantiacus cbh1 7 S. cerevisiae NR NR NR 65 NRHumicolainsolens

cbhII 6 S. cerevisiae NR T1/2: 95 min at63°C

9 57 NR Heinzelman et al., 2009

H. insolens Hicel6C 6 P. pastoris NR After incubation at60°C for 1 h, theenzyme retainedgreater than 90% ofits initial activity

6.5 70 NR Xu et al., 2015

Myceliophthorathermophila

eg7a 7 P. pastorisX-33

4.76 Retained more than40 % attemperatures up to80 °C for 8 h ofincubation

5 60 65.0 Karnaouri et al., 2014

TalaromycesemersoniiCBS394.64

TeEgl5A 5 P. pastorisGS115

NR Enzyme remainedmore than 70 % ofthe maximalactivity at 80–95°C

4.5 NR 36.8 Wang et al., 2014

Table 2 Biochemical properties of recombinant cellulases of thermophilic moulds

Bijender Singh, Anju Bala, Seema Dahiya and T. Satyanarayana 51

that enzyme had two domains, a chemical action domain andother one was cellulose binding domain which was covalentlyjoined by a 33- amino acid sequences. Mackenzie . (1998)found that endoglucnasae was a .Cel7A catalyst (cellobiohydrolases) ofshowed more thermal stability than Cel7A onswollen polyose, and had higher ability to degrade pre-treatedcorn fodder at 65˚C (Momeni ., 2014). The structure ofendoglucnase from and was similar buttheir optimum pH values were different (Kleywegt .,1997), their pH optima were totally different at 7.5 and 4.5(Sch lein, 1997).

.,2010) that showed optimal activity at pH 6.0 and 40˚C. Themolecular mass of the enzyme was 57 kDa. The enzymeshowed positive activity against cellobiose and inhibited bymetal ions such as Zn ,Al , Cu , Fe . Using p-nitrophenyl-D-glucopyranoside (pNPG) as the substrate,

mg and Km of0.16 mM (Benoliel ., 2010). Cellobiose dehydrogenases(CDH) of and (Canevascini .,1991; Coudray ., 1982; Schou ., 1998) were clonedand expressed. The recombinant enzymes had molecularmasses of 92 and 95 kDa. The optimum pH and temperaturefor the activity of was 7.5 and 60 C. For that of

, optimal pH was 4.0 (Schou ., 1998;Subramaniam ., 1999; Igarashi ., 1999).

Structure and sequencing analysis of cellobiosedehydrogenase of showed that the enzyme had3 different domains: catalytic domain with ketone end, amiddle heme domain and cellulose binding domain ofcellulase (Igarashi ., 1999). Glycosylation was dependenton the strain and culture conditions (Bernstein ., 1977).Notably, once a gene coding for cellulase fromwas expressed in , enzyme contained large number ofN- glycosylation cites in the enzyme active site (Basha andPalanivelu, 1998). Murray . (2004) isolated cel3a from

and expressed inthat showed high thermal stability (T of 62 min at

65˚C). Enzyme had 71.5 C as the optimal temperature withV of 512 1U/ mg and K of 0.254 mM. wasused for the over expression of

IFO9748 (Hong ., 2007). Theenzyme belongs to family GH3. Recombinant enzyme washighly thermostable and retained 70 % activity after 1 hexposure at 60˚C, but the optimum temperature for enzymeactivity was 70 ˚C. Recombinant enzyme was also stable in abroad pH range from 3.0-8.0 with optimal activity at pH 5.0.Cellobiose was found as the best substrate for the enzyme(Hong ., 2007). By using mathematical modelling, it wasfound that CBH II from expressed in

was an extremely stable cellulase with a relativemolecular mass of 55 kDa (Heinzelman ., 2009). A gene(TeEgl5A) from thermophilic mouldencoding nase was functionally expressedin (Wang ., 2014). The recombinantenzyme exhibited optimal activity at 90˚Cand pH4.5 andwastolerant to a wide range of pH 1.0-10.0. The recombinantenzyme showed a grater resistance to metal ions and

surfactants, and it was found that the enzyme displayed higheractivi

idic bonds such as laminarian, lichenan, CMC-Na, birchwood xylan anddisplayed a higher efficiency in reducing medium viscositythan the commercial enzyme Ultraflo XL from novozyme(Wang ., 2014).

Another cellulase enzyme gene form Y1(Hicel6C) was expressed in (Xu ., 2015). Therecombinant enzyme had optimal activity at neutral pH and70˚C. The enzyme was different from those of thermophilicmoulds, being alkali- tolerant retaining more than 98.0, 61.2and 27.6 % of activity at pH 8.0, 9.0, and 10.0, respectively

, the enzyme had 1.29 mg/mol/min/mg as K and V values, respectively.

HiCel6C enzyme washigh

nan as compared to laminarin (1,showed capacity to degrade

the glycosidic linkages interior of cellooligosaccrides, thus agood endo-cleaving catalyst in nature (Xu ., 2015).Cellobiohydrolase II (CBH II) ofCT2 was expressed in under the inducible AOXIpromoter and produced 1.2 mg/ml of pure enzyme (Liu .,2005). The recombinant enzyme was purified using saltprecipitation and ion exchange quick flow chromatography.The molecular mass determination was done by using SDS-PAGE and it was exactly the same as the nativecellobiohydrolase II from CT2 (67kDa).The enzyme was optimally active at 50˚C and pH 4.0 5.0.Enzyme was thermostable that retained 50% of the originalactivity for 30 min at 70˚C. The structure analysis ofcellobiohydrolase IB of the fungus showed thatthis enzyme was a oversized glycoprotein with single domainand that belongs to family GH7(Grassick ., 2004).

The enzyme industry has developed very rapidly in the lastfew decades. The total estimated market value of the enzymeindustry was $ 5.0 billion in 2015, which is expected toincrease to $ 6.3 billion in 2021 at a compound annual growthr a t e ( C A G R ) o f 4 . 7 % f o r 2 0 1 6 - 2 0 2 1[http://www.bccresearch.com]. Cellulases (~ 15 %) also havea great impact on enzyme industry and it occupies thirdimportant enzyme in industrial applications. The other twoimportant enzymes are amylase ( ~ 25%) and protease (~ 18%). Microorganisms are the important sources of cellulases(Bhat, 2000; Kuhad ., 2010). Cellulase production fromthermophilic moulds received attention because of theirutilization of all types of substrates mainly lignocellulosicbiomass (Kaur and Satyanaryana, 2004; Kuhad ., 2010).Now-a-days, energy production by using agriculturalresidues is gaining attention of many industries as it is a cheapand renewable. Use of inexhaustible energy source isencouraged because these sources can generate 10-14%world's energy supply. Apart from various chemicals andphysical methods used for degradation of cellulosic biomass,

et al

Humicola griseaH. jecorina

et alH. insolens T. reesei

et al

et al

et alS. thermophile H. insolens et al

et al et al

H. insolens S.thermophile et al

et al et al

S. thermophile

et alet alT. emersonii

T. reesei

et alTalalaromyces emersonii Trichodermareesei

Pichia pastoris

Thermoascus aurnaticus et al

et alHumicola insolens S.

cerevisiaeet al

Talaromyces emersonii

Pichia pastoris et al

et alHumicola insolens

P. pastoris et al

et alChaetomium thermophilum

P. pastoriset al

C. thermophilum

T. emersonii

et al

et al

et al

polypeptide with β-sheets

The recombinant β-glucosidase wassuccessfully expressed in brewer's yeast (Benoliel

β-recombinant

enzyme showed Vmax of 6.72 μmol min

β-glucosidase from

endo-1,4-β-gluca

ty on substrates which had β-1,4-glycosidic bonds andβ-1,3-glycos

barley β-glucan. The enzyme

By using β-glucan as a substrate mland 752 μ

specific to degrade β-1,4-glycosidicbonds and showed enzyme activity using β-glucan,CMC-Na, liche 3-β-glucan).It was also found that HiCel6C

βsandwich structure

ü

.

2+ 3+ 2+ 2

-1 -1

o

o1/2

max i

m max

A P P L I C A T I O N S O F C E L L U L A S E S O FTHERMOPHILIC MOULDS

Production, characteristics and potential applications of the cellulolytic enzymes of thermophilic moulds52

the use of enzymes of thermophilic microorganisms is beingencouraged. The enzyme-based degradation of biomassfinds applications in various industries ( ) [Bergquist

., 2004; Bamforth, 2009; Kuhad ., 2010].

Cellulases occupy a place in food industry.Cellulases are used as macerating enzymes along withxylanases and pectinases for the clarification and improvedextraction of juices from fruits and vegetables. The stability ofjuices also improves when macerating enzymes are used forthe extraction along with higher yields of juices (Kaur .,2004). Kaur . (2004) studied the effect of mixture ofpectinase, cellulase, xylanase enzymes fromon the yield of various fruit juices such as banana, grape andapple and found that the yields of juices increased ascompared to when pectinase was used alone for extraction(Kaur ., 2004). Cellulases, xylanases and pectinases areused in food biotechnology for improving the properties offruits and vegetables (Bhat, 2000). The enzymes are also usedto improve the texture of nectars and purees from tropicalfruits (mango, pears, apples, apricot, papaya, peach and plum)[Sukumaran ., 2005; Bhat, 2000]. Citrus fruit propertiessuch as texture, flavour, and aroma of fruits were alsoimproved by using

., 2007). Use of cellulase enzymesdecreased the viscosity of nectars and purees rapidly ascompared to other methods (Grassin ., 1996). In thepresent scenario, the use of cellulases in food industry isincreasing along with other macerating enzymes as they canbe used for all type of fruits and vegetables without anysignificant loss of yields and improvement in the digestion ofanimals (Dourado ., 2002).

The use of cellulases andhemicellulases in feed industry draw considerable attentionof researchers as they have potential to improve the feed valueof animal diets, thus, leading to improved efficiency ofanimals (Dhiman ., 2002). Pre-treatment of agriculturalresidues, which are used as animal feeds, led to improvementin digestibility (Godfrey and West, 1996). The enzymes notonly improved the feed value but also eliminated the anti-nutritional factors. Dietary fibre consists of various non-

starch poylsaccharides such as inulin, lignin, dextrins,waxes and oligosaccharides (Dhiman ., 2002). By

the action of cellulases, anti-nutritional factors are removedleading to improvement in digestion and health of animals.Cellulases not only improve the silage production but alsoincrease the digestibility of grasses. Cellulases can be used toimprove the digestibility of silage production for cattlefeeding making the availability of total digestible nutrientsand energy with low water-soluble carbohydrates (Lewis

., 1996). Enzyme cocktail comprising of cellulase,hemicellulase, and pectinase have been used in improving thenutritional quality of the forages (Lewis ., 1996). Bothcellulases and hemicellulases are responsible for dehulling ofcereal grains, partial hydrolysis of lignocellulosic biomass, -glucans hydrolysis and better emulsification of feed materialsand thus, improving the nutritional value of animal feeds(Cowan, 1996).

World faced a worldwide oil crisis in 1970s that ledto increased attention on the use of cellulases in bio-fuelindustry. The enzyme is used to degrade the cellulosic wastesinto fermentable sugars, which are fermented to bio-ethanol.The purpose of this process is to reduce the burden of oilindustry by reducing the import of oil. This will improve thequality of air by reducing the emission of gases in the air.Bioethanol is the most common and eco-friendly renewablefuel produced from lignocelluosic biomass by thermostableenzymes of thermophilic moulds by simultaneousliquefaction, saccharification and fermentation (Lynd2005). For efficient bioconversion of lignocellulosicbiomass, a strategy of efficient saccharification usingcellulolytic enzymes is required. Thermostability is animportant and desirable feature for cellulases as thesaccharification of cellulose is faster at higher temperatures.Mesophilic has high endo and exoglucanaseactivities therefore, it haslimited utility in the hydrolysis of cellulose. Therefore,thermophiles could be suitable substitutes for this fungus.Thermophilic moulds such as(Kaur , 2004; Berka , 2011; Singh 2016; Singh2016) (Gomes , 2000; Jain

., 2015) and(Berka ., 2011) have efficient enzymatic machinery forthe hydrolysis of lignocellulosic materials. These microbeshave, therefore, been proposed as good candidates for theconversion of lignocellulosic residues to sugars, therefore,have a great potential for applicability in bioethanolindustries (Berka , 2011). The crude enzymes from

BJAMDU5 resulted in the hydrolysis of wastetea cup paper and rice straw with a sugar yield of 578.12 and421.79 mg/g substrate, respectively (Bala and Singh, 2016).

and werecultivated on various substrates for producing glycosidehydrolases (McClendon ., (2012). Crude culture filtratesused in the saccharification of ionic liquid pre-treated switchgrass ( ) revealed thatenzymes released more sugars (glucose) thanenzymes (McClendon ., 2012).

Cellulases are utilized in textile wetprocess, particularly in finishing of cellulose-based textiles,

Table 3

Food industry:

Animal feed industry:

Biofuels:

Textiles industry:

et al et al

et alet al

S. thermophile

et al

et al

et al

et al

et al

et al

et al

etal

et al

et al.,

Trichoderma

Sporotrichum thermophileet al. et al. et al.,, Thermoascus aurantiacus et al. et

al Scytalidium thermophillum Thielavia terrestriset al

et al. S.thermophile

Thermoascus aurantiacus Thielavia terrestris

et al

Panicum virgatum T. aurantiacusT. terrestris

et al

enzymes such as pectinases and β-glucosidases (Rai

β-glucan,

with lower β-glucosidase levels,

Industry Applications ReferencesAgriculture Plant pathogen and disease control, enhanced seed germination, rapid

plant growth and flowering, increased crop yields, reduced dependenceon mineral fertilizers, preservation of high quality fodder.

Bhat, 2000; Sukumaran et al.,2005; Singh et al., 2007;Karmakar and Ray 2011

Biofuels Bioethanol production, production of fuel oxygenates, bio-butanolproduction, H2 production, conversion of cellulosic materials toethanol, other solvents, organic acids, and lipids.

Sukumaran et al., 2005; Kuhadet al ., 2011

Detergent Cellulase-based detergents, superior cleaning action without damagingfibers. Removal of rough protuberances in cotton fabrics, improvedcolour brightness and dirt removal, anti- redeposition of ink particles

Sukumaran et al., 2005; Singhet al., 2007; Karmakar andRay 2011

Fermentation Production of the wine, beer and liquor, activity of cellulase incomposition with α-amylase and glucoamylase has been selected toachieve a higher ethanol yield in the distillate, by decreasing theconcentrations of methanol, propanol, isobutanol and isoamyl & amylalcohols concentration, improved malting and mashing; improvedpressing and color extraction of grapes; improved aroma of wines;improved primary fermentation and quality of beer; improved viscosityand filterability of wort; improved must clarification in wineproduction; improved filtration rate and wine stability

Bhat , 2000; Sukumaran et al.,2005; Kuhad et al., 2011

Food and feedprocesses

Pretreatment of agricultural silage and grain feed, enhancement of thedigestibility of grasses, improvement of yields in starch and proteinextraction, decreased viscosity of the nectars from fruits, used in theimprovement of the bakery products, improved feed digestion andabsorption, increases the nutritive value of the feed, release of theantioxidants from fruit and vegetable pomace, clarification of fruitjuices, improved texture, aroma and flavour, improved nutritionalquality of animal feed of the fruit juices, improved texture and qualityof bakery products, production of single cell protein

Rai et al., 2007; Karmakarand Ray, 2011

Textiles andBiopolymers synthesis

Mercerization of cotton, wool scouring, biostoning of jeans,biopolishing of textile fibers; pretreatment of bast fibers including jute,flax and ramie, softening of garments; improved stability of cellulosicfabrics; removal of excess dye from fabrics; restoration of colourbrightness

Kaur et al., 2004; Kuhad et al.,2011

Agro and plantswaste treatment

Application in biogas production unit, reduction of agricultural andmunicipal solid waste residues, production of the fermentable sugarsfor biofuels and useful products, reduce environmental production,bioconversion of lignocelluloses.

Karmakar and Ray, 2011;Kuhad et al., 2011

Table 3. Potential biotechnological applications of cellulolyticenzymes of thermophilic moulds

Bijender Singh, Anju Bala, Seema Dahiya and T. Satyanarayana 53

with the goal of improved look (Hebeish andAbraham, 2007;Karmakar and Ray, 2011). Normally the thermophiliccellulases are utilized in the stone laundry of jeans to makethem look light and in biopolishing of cotton (Kuhad2011). These enzymes are also mixed with detergents forreducing the discolouration and fuzzing effects resulted byrepeated washing (Kuhad , 2011). Due to recurrentlaundry, most cotton blended clothes tend to become flossyand less attractive, therefore, cellulases present in thesedetergents will take away these microfibrils and restore asleek surface and original color to the clothes. Use ofcellulases for treatment of raw cotton fibers i.e. non plain-woven materials improved the texture of fibres (Singh .,2007). Use of cellulases together with lipase within thedetergents could be a newer innovation in the field (Singh

, 2007). Cellulase preparations capable of modifyingpolyose fibrils will improve color brightness, feel, and dirtremoval from the cotton mix clothes. The economicapplication of basic cellulases as a possible detergent additiveis being actively pursued (Singh ., 2007; Sukumaran

., 2005). Nowadays, liquid detergents containing anionic ornon-ionic wetting agent, acid or a soluble salt, protease,cellulase, and a mix of propanediol and boric acid or its by-product are used for improving the efficiency of cellulases.The cellulases are useful in the removal of roughprotuberances in glossier and brighter-coloured materials(Karmakar and Ray, 2011).

Various preparations consisting of mixtures ofcellulases, hemicellulases, and pectinases have potentialapplications in agriculture for enhancing the growth of crops(Bhat, 2000). Historically straw incorporation is taken intoaccount as a vital strategy to enhance soil quality and cut backdependence on mineral fertilizers (Ortiz Escobar and Hue,2008, Tejada , 2008). Microbial cellulases play a vitalrole in fermentation processes to supply alcoholic beveragestogether with beers and wines (Singh , 2007).Macerating enzymes conjointly improve pressability, settlingand juice yields of grapes used for wine fermentation. Avariety of economic catalyst preparations are currently usedin wine trade. Vital and consistent enhancements in grapepressability, subsidence rate, and total juice yield wereachieved through a mixture of macerating enzymes. Suchenhancements were noticeable solely with an accuratebalance of pectinases, cellulases and hemicellulases. Avariety of improved enzymes like cellulase and pectinase thatmight be exogenously added is expected to boost theproductivity of the existing production processes in future(Bamforth, 2009).

Thermophilic moulds are known to secrete cellulolyticenzymes in submerged as well as solid state fermentations.Cellulases of thermophilic moulds have desirable properties,thus, better suited for industrial applications. Cellulases ofthermophilic moulds have been cloned and expressed indifferent hosts and characterized. Cellulases have beenutilized in saccharifying cellulosics, improving food and feednutrition and other industries. Furthermore, the diversity ofthermophilic moulds is less explored as compared to

mesophiles. Further research efforts are therefore called forexploring the diversity of thermophilic moulds, studyingstructure - function aspects of cellulases and amelioratingcatalytic activities of cellulases by site directed mutagenesisand directed evolution.

The authors are grateful to Council of Scientific and IndustrialResearch, Government of India, New Delhi (Grant No.38(1370)/13/EMR-II), Haryana State Council for Scienceand Technology (Grant No. HSCST/R&D/2017/62), andUniversity Grants Commission (Faculty Fellowship) and toIUSSTF (to TS) for the financial assistance while writing thisreview.

Bajaj, B.K., Sharma, M. and Rao, R.S. 2014. Agriculturalresidues for production of cellulase from

LAR5 and its applicationfor saccharification of rice straw.

: 1454-1460.

Bala, A. and Singh, B. 2016. Cost-effective production ofbiotechnologically important hydrolytic enzymesby .

: 181-191.

Bamforth, C.W. 2009. Current perspectives on the role ofenzymes in brewing. : 353-357.

Basha, S.Y. and Palanivelu, P. 1998. Enhancement in activityof an invertase from the thermophilic fungus

by exogenous proteins.: 603-605.

Begum, F., Absar, N. and Alam, M.S. 2009. Purification andcharacterization of extracellular cellulase from

ITCC-4857.01. : 1646-1651.

Benoliel, B., Poças-Fonseca, M.J., Torres, F.A. and deMor ae s , L. M. 20 10 . Pur i f ic a t i on a ndcharacterization of expression of a gl

varin .

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Berka, R.M., Grigoriev, I.V., Otillar, R., Salamov, A.,Grimwood, J., Reid, I., Ishmael, N., John, T.,Darmond, C., Moisan, M.C., Henrissat, B.,Coutinho, P.M., Lombard, V., Natviq, D.O.,Lindquist, E., Schmutz, J., Lucas, S., Harris, P.,Powlowski, J., Bellemare, A., Taylor, D., Butler, G.,de Vriies, R.P., Allijn, I.E., van den Brink, J.,Ushinsky, S., Storms, R., Powell, A.J., Paulsen, I.T.,Elbourne, L.D., Baker, S.E., Maqnuson, J.,Laboissiere, S., Clutterbuck, A.J., Martinez, D.,Woqulis, M., de Leon,A.L., Rey, M.W.and Tsanq,A.

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