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Expression pattern of Trichoderma cellulases
under different carbon sources
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Pak. J. Bot ., 42(4): 2895-2902, 2010.
EXPRESSION PATTERN OF TRICHODERMA CELLULASES
UNDER DIFFERENT CARBON SOURCES

NIGHAT ASLAM1, MUNIR A SHIEKH1, MUHAMMAD ASHRAF2 AND AMER JAMIL1*

1Molecular Biochemistry Lab., Departme nt of Chemistry and Biochemistry,
University of Agriculture, Faisalabad- 38040. Pakistan.
2Department of Botany, University of Ag riculture, Faisalabad. 38040. Pakistan.
2Second Affiliation: King Saud University, Riyadh, Saudi Arabia.

Abstract

Production of easily fermentable low-cost sugars demands for an economic and less expensive
method of enzymatic hydrolysis of cellulosic biomass. It is highly desirable to compare various
fermentation media with different carbon sources for cellulase production. Trichoderma harzianum
was grown on different carbon sources and monitored for cellulase production. Glucose-grown
cultures of T. harzianum showed high amount of mycelial growth but no yield of cellulase enzyme.
Cellulase expression was also studied herein by comparing the cellulase activities using soluble and
insoluble cellulosic carbon sources in the growth media in order to obtain less expensive
fermentation media. Outcome of the research will be helpful in the development of low cost system
for production of cellulose.

Introduction

Fungi like Trichoderma secrete a large number and a variety of enzymes that can act
on the polycassharides found in plant cell walls. These enzymes include cellulases,
hemicellulases, pectinases, esterases, oxidoreductases and proteases (Chandra et al., 2010).
The polysaccharides especially cellulose s and hemicelluloses are very cheap and
easily available as wastes from industries like paper and pulp, agricu lture, food and feed
and municipal. In developing countries these wastes are not been discarded or treated
properly and become the major cause of environmental po llution (Dashtban et al., 2009).
Cellulose is a linear biopolymer of glucopyranose-molecules, connected by β-1,4-
glycosidic b onds. Cellulase enzymes, which can hy drolyze cellulose forming glucose and
other commodity chemic als, can be divided into three types: endoglucanase (endo-1,4- β-
D-glucanase, EG, EC 3.2.1.4); cellobiohydrolase or exoglucanase (exo-1,4- β-D-
glucanase, CBH, EC 3.2.1.91) and β-glucosidase (1, 4- β- D-glucosidase, BG, EC
3.2.1.21) (Li et al., 2006; Gao et al., 2008; Ahmed et al., 2009b).
Some species of filament ous fungi secrete more than two hundred glycosyl
hydrolases alone (Nagendran et al., 2009). Trichoderma species has been used for a long
time for industrial enzyme production and as an important model system for studying
cellulosic and lignocellulosic degradation (Nevalainen et al ., 1994). Its enzymes have
important applications in starch processing, pulp and paper industry , extraction of fruit
and vegetable juices, malting and brewin g, animal feed production and alcohol
fermentation. Many of the extracellular enzymes of T. reesei have been biochemically
characterized (Stricker et al., 2008; Nagendran et al., 2009; Sun et al., 2010).
In one of previous study at our lab, three cellulases, exoglucanase (EXG),
endoglucanase (EG) and β-glucosidase (BGL) were partially purified from T. harzianum
(Ahmed et al., 2009a). The opti mal pH, temperature and incubation time for cellulases
production were determined. In this study we report the time course effect of different
carbon sources like CMC (carbo xymethylcellulose), lactose, sigma cellulose, birchwood
xylan, wheat bran and alkali treated corn cobs and glucose on the pr oduction of cellulases
by Trichoderma harzianum .
*Corresponding author: amerjamil@yahoo.com

NIGHAT ASLAM ET AL .,
2896
Materials and Methods

Chemicals: Carboxymethylcellulose (CMC), glucose, Sigma cell and lactose were from
Sigma Chemical Co., USA. Corn cobs and wh eat bran were gift from Fermentation Lab
University of Agriculture, Faisalabad, Paki stan. All the other chemicals used were of
analytical grade unless otherwise stated.

Fungal strain and culture conditions: Trichoderma harzianum (E-58) was maintained
on agar slants containing (g/L) Trisodium citrate, 0.5; KH 2PO 4, 0.5; NH 4NO 3, 0.2;
(NH 4)2SO 4, 0.4; MgSO 4, 0.02, peptone, 0.1; yeast extrac t 0.2; glucose 0.2; agar 2.5.
Inoculated slants were incubated for 5 days and spores were transferre d to inoculum
containing Vogel’s medium (Vogel, 1956) containing glass beads for uniform
suspension. The fungal growth for enzyme production was carried out in the Vogel’s
medium containing 1% any of the carbon source. Trace element solution (Citric acid.
H2O, 5g; ZnSO 4 7H 2O, 5 g; Fe(NH 4)2(SO 4)2 6 H 2O, 1 g; CuSO 4 5H 2O, 250 mg; MnSO 4
H2O, 50 mg; H 3BO 3, 50mg; Na 2MoO 42H 2O, 50 mg and H 2O up to 100 mL) was added
for optimal growth and enzyme production. Th e flasks were incubated for 5 days in a
shaking incubator at 180 rpm at 28 °C (Aslam et al., 2004).

Enzyme assays: After each 24 hours cr ude cultures from different flas ks containing
different carbon sources were withdrawn and ch ecked for the activity of cellulases. The
carbon sources used were lactose, CMC, ce llulose (sigma cell), bi rchwood xylan, wheat
bran and alkali treated corn cobs. Three fundamental cellulase enzymes Endoglucanase,
Exoglucanase and β-glucosidase were checked for their activity in the crude cultu re filtrate
of all above mentioned carbon sources. In every assay DNS (Dinitorsalycili c acid) method
(Shamala & Sereekanth, 1985) was employed to check the ac tivity of enzymes. Enzyme
(0.2 mL crude culture filterate) was incuba ted with 1.8 mL of 1% substrate in 0.2 M acetate
buffer at 50°C for 30 minute s. The DNS (3 mL) was added to stop the reaction and boiled
for 10 minutes in boiling water bath. Afte r cooling the reaction, OD was noted at 575 nm.
The enzyme activities were determined by comparing the am ount of product formed.

Protein estimation: Protein was estimated using Bradfo rd’s reagent (Bradford, 1976).
To 1.5 ml Bradford reagent 50 µL prot ein (crude enzyme) was added and OD was
checked at 595 nm after 10 minutes.

Results and Discussion

Trichoderma harzianum was grown in Vogel’s media having trace element solution
and Tween 80. It was observed that using trac e element solution the mycelial growth was
very high and enzyme produc tion reaches its maximum with in 48 hours. Tween 80 was
used to inhibit the pellet formation so as to increase protein concentration and cellulase
activity (Domingues et al., 2000). Furthermore it also he lps in increasing the production
of cellulases (Reese & Maguire, 1969). Regula tion of expression of three cellulases was
monitored in the fungal species. Carbon sources like Carboxymethylcellulose (CMC), Sigma cellulose, lactose, alkali treated corn cobs, wheat bran and glucose were used to
investigate their influence on the cellulas es production. Vogel’s media (Vogel, 1956;
Montenecourt & Eveleigh, 1977) was used for the present study. Repression of the enzymes was noticed during growth on glucose. When the glucose was added after 24 or
48 hours the cellulase level remains consta nt. Literature survey also shows that
expression of a large majority of the cellulase genes that have been studied in H. jecorina
and other filamentous fungi is believed to be induced du ring growth on cellulosic
substrates and does not occu r during growth on glucose (Messner & Kubicek, 1991;

TRICHODERMA CELLULASES UNDER DIFFERENT CARBON SOURCES
2897
Cullen & Kerston, 1992; Kubicek et al., 2009). Protein was de termined using Bradford
method (Bradford, 1976). Figure 1 show s the enzyme activities of exoglucanase,
endoglucanase and β-glucosidase using CMC as a substrate. Endoglucanase showed the
higher activity on CMC as co mpared to exoglucanase and β-glucosidase. This explains
that CMC is a specific substrate of endoglucanase (Xiao et al ., 2005). The enzyme
activities of exoglucanase, endoglucanase and β-glucosidase using sigma cell as a
substrate were very low during first three days having lowe st activity of endoglucanase,
but with time the act ivity of endoglucanase increase d (Fig. 2). Since sigma cell
(cellulose) is not easy to be broken down, it s activity was less as compared to CMC.
Figure 3 shows the enzyme acti vity using lactose as a carbon source in the growth media.
Lactose is a disaccharide and is readily us ed by fungus to induce cellulases. Kubicek
(2009) studied the induction of cellulases using lactose and re vealed that the induction of
cellulase transcription by this disaccharide required simultaneous degradation of the D-
galactose moiety of lactose and the alternative reductive D -galactose catabolic pathway.
However, as the experiments were performe d on the crude cultures and cellulose needs
synergistic action of different enzymes so other enzymes may be involved in giving the cellulase activity. Addition of glucose to the growth media c ontaining cellulosic
meterials results in negligible increase in the formation of cellulases (Figs. 6 and 7).
Cellulase formation was also mo nitered in the culture filtrate which had only glucose as a
carbon sources in the medium, which shows al most zero activity but gave large amount
of mycelial mass. As glucose is the end produc t of cellulose degradation by cellulases, so
the presence of glucose in the medi um shows the end product inhibition
(Muthuvelayudham & Viruthagiri, 2006). Szijarto et al., (2004) also found similar results
while monitoring the cellulase producti on in the glucose-gr own cultures of T. reesei Rut-
C30 and under pulse add itions of Solka-floc. Cullen & Ke rston (1992) e xplained in the
same way that enzyme synthe sis is inhibited by end-product like glucose. However,
Messner & Kubicek (1991) detected low levels of T. reesei exoglucanase (CBHII) in
glucose medium. Rapid growth in the mycelia was noticed with glucose in the media but
with lactose, CMC, sigma cell, wheat bran and corn cobs, there was slow mycelial
growth. Induction in Trichoderma has been shown to occur at the transcriptional level.
Cellulases are induced in most of fungi only when cellulose or an inducer exists (Suto &
Tomita, 2001). It has also be en suggested that low level of expression of cellulases
mediate the induction by pr oducing soluble compounds th at enter the cell and cause
induction (Teeri et al., 1987; Kubicek et al., 2009). Trichoderma needs low level (basal
cellulases) for the induction of cellulases. Thes e cellulases (basal cellu lases) would digest
cellulose and release oligosaccharides that en ter into the cel l and trigger expression of
cellulases. Carle-Urioste et al., (1997) revealed that the basal expression of the cellulase
was required for induction of its own transcripts by cellulo se. Figure 4 and 5 show the
enzyme activities using insoluble and relati vely complex cellulosic substrate i.e., wheat
bran and alkali treated corn cobs in the growth media. As shown in the figures the enzyme activities were low as compared to the above discussed media, and increase in
activities with time was also lo w. This explains that as whea t bran and alkali treated corn
cobs are complex substrates so the enzymes found difficult to penetrate into these substrates. Liming & Xueliang (2004) used corn cob residues fo r the production of
cellulases using Trichoderma reesei ZU-02. They found that the cellulase production was
the same with corn cobs and commercially avai lable purified cellulose. In our case, corn
cobs were not finely ground which results in lower enzy me activity. As purified
celluloses are very expensive therefore by usin g the finely ground corn cobs, cellulase
yield can be increased.

NIGHAT ASLAM ET AL .,
2898
00.150.30.450.60.750.91.05
24 48 72 96 120
hours of incubation umoles/min/mLexoglucanase
Endoglucanase
beta glucosidase

Fig. 1. Cellulase activity in the culture filtrate of T. harzianum with CMC as a carbon source. DNS
assay was used to test the reducing sugars formed during hydrolysis. Avicel, CMC and salicin were
used as a substrate in the assays of exoglucanase, endoglucanase and β-glucosidase, respectively.
No enzyme control was used as a blank.

00.020.040.060.080.10.120.140.160.18
24 48 72 96 120sigm a cell
hours of incubation umoles /min/mLexoglucanase
Endoglucanase
beta glucosidase

Fig. 2. Cellulase activity in the culture filtrate of T. harzianum with sigma cell as a carbon source.
DNS assay was used to test the reducing sugars formed during hydrolysis Avicel, CMC and salicin
were used as a substrate in the assa ys of exoglucanase, endoglucanase and β-glucosidase
respectively. No enzyme control was used as a blank.
Hours of incubation µmoles/min/mL µmoles/min/mL
Hours of incubation

TRICHODERMA CELLULASES UNDER DIFFERENT CARBON SOURCES
2899
00.10.20.30.40.50.6
24 48 72 96 120
hours of incubation umoles/m in/mL
exoglucanase
Endoglucanase
beta glucosidase

Fig. 3. Cellulase activity in the culture filtrate of T. harzianum with lactose as a carbon source
DNS assay was used to test the reducing sugars formed during hydrolysis. Avicel, CMC and salicin
were used as a substrate in the assa ys of exoglucanase, endoglucanase and β-glucosidase
respectively. No enzyme control was used as a blank.

00.010.020.030.040.050.060.070.080.09
24 48 72 96 120
hoursumoles/min/mLexoglucanase
Endoglucanase
beta glucosidase

Fig. 4. Cellulase activities in the culture filtrate of T. harzianum with wheat bran as a carbon
source. DNS assay was used to test the reducing sugars formed during hydrolysis. Avicel, CMC
and salicin were used as a substrate in the assays of exoglucanase, endoglucanase and β-
glucosidase respectively. No enzyme control was used as a blank
Hours of incubation µmoles/min/mL
Hours µmoles/min/mL

NIGHAT ASLAM ET AL .,
2900
00.020.040.060.080.10.12
24 48 72 96 120
hours
exoglucanase
Endoglucanase
beta glucosidase

Fig. 5. Cellulase activities in the culture filtrate of T. harzianum with corn cobs as a carbon source.
DNS assay was used to test the reducing sugars formed during hydrolysis. Avicel, CMC and salicin
were used as a substrate in the assays of exoglucanase, endoglucanase and β-glucosidase
respectively. No enzyme control was used as a blank.
00.050.10.150.20.25
02 4 4 8 7 2 9 6 1 2 0
Time (hours)umoles/min/mLexoglucanase
Endoglucanase
beta glucosidase

Fig. 6. Cellulase assay on the media containing sigma cell with addition of glucose. Glucose (1%)
was added after 48 hours of incubation in the media containing sigma cellulose as carbon source.
Cellulase assay was performed after every 24 hours to monitor the presence of cellulase enzyme.

00.10.20.30.40.50.60.70.8
24 48 72 96 120
hoursumoles/min/mLexoglucanase
Endoglucanase
beta glucosidase

Fig. 7. Cellulase assay on the media containing CMC with addition of glucose. Glucose (1%) was
added after 48 hours of incubation in the media containing CMC as carbon source. Cellulase assay
was performed after every 24 hours to monitor the presence of cellulase enzyme.

µmoles/min/mL
Hours
Time (hours) Umoles/min/mL µmoles/min/mL
Hours

TRICHODERMA CELLULASES UNDER DIFFERENT CARBON SOURCES
2901
References

Ahmed, S., A. Bashir, H. Saleem, M. Saadia and A. Jamil. 2009a. Production and purification of
cellulose degrading enzymes from a filamentous fungus Trichoderma harzianum . Pak. J. Bot .,
41: 1411-1419.
Ahmed, S., S. Riaz and A. Jamil. 2009b. Molecular cloning of xylanases: an overview. Appl.
Microbiol. Biot ., 84: 19-35.
Aslam, N., F. Latif, S. Ahmed and A. Jamil. 20 04. Molecular Cloning of ß Glucosidase gene from
Trichoderma harzianum . Biotechnology , 3: 63-66.
Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of
protein utilizing the principle of protein–dye binding. Anal Biochem ., 248-54.
Carle-Urioste, J.C., J. Escobar-Vera, S. El-Gogary, F. Henrique-Silva, E. Torigoi, O. Crivellaro, A.
Herrera-Estrella and H. El-Dorry. 1997. Cellulase Induction in Trichoderma reesei by
Cellulose Requires Its Own Basal Expression. J. Boil. Chem ., 272: 10169-10174.
Chandra, M., A. Kalra, P.K. Sharma, H. Kumar and R.S. Sangwan. 2010. Optimization of
cellulases production by Trichoderma citrinoviride on marc of Artemisia annua and its
application for bioconversion process. Biomass Bioenerg. , Article in press.
Cullen, D and P. Kersten. 1992. Fungal enzymes for lignocellulose degradation. In: Applied
Molecular Genetics of Filamentous Fungi . Chapman & Hall, New York, chapter 4.
Dashtban, M., H. Schraft and W. Qin. 2009. Fungal bioconversion of lignocellulosic residues;
Opportunities & perspectives. Int. J. Biol. Sci ., 5: 578-595.
Domingues, F.C., J.A. Queiroz, J. M.S. Cabral and L.P. Fonseca. 2000. The influence of culture
conditions on mycelial structure and cellulase production by Trichoderma reesei RUT C-30.
Enzyme Microb. Technol ., 26: 394-401.
Foreman, P.K., D. Brown, L. Dankmeyer, R. Dean, S. Diener, N.S. Dunn-Coleman, F.
Goedegebuur, T.D. Houfek, G.J. England, A.S. Kelley, H.J. Meerman, T. Mitchell, C. Mitchinson, H.A. Olivares, P.J. Teunissen, J. Yao and M. Ward. 2003. Transcriptional regulation of biomass-degrading enzymes in the filamentous fungus Trichoderma reesei . J.
Biol. Chem ., 278: 31988-31997.
Gao, J., H. Weng, D. Zhu, M. Yuan, F. Guan and Yu Xi. 2008. Production and characterization of
cellulolytic enzymes from the thermoacidophilic fungal Aspergillus terreus M11 under
solidstate cultivation of corn stover. Bioresource Technol ., 99: 7623-7629.
Kubicek, C.P., M. Mikus, A. Schuster, M. Schmol l and B. Seiboth. 2009. Metabolic engineering
strategies for the improvement of cellulase production by Hypocrea jecorina. Biotechnol.
Biofuels , 2: 19.
Li, Y.H., M. Ding, J. Wang, G. J. Xu and F. Zhao. 2006. A novel thermoacidophilic endoglucanase,
Ba-EGA, from a new cellulose degrading bacterium, Bacillus sp. AC-1. Appl. Microbiol.
Biot., 70: 430-436.
Liming, X. and S. Xueliang. 2004. High-yield cellulase production by Trichoderma reesei. ZU-02
on corn cob residue. Bioresource Technol ., 91: 259-262.
Messner, R. and C.P. Kubicek. 1991. Carbon Source Control of Cellobiohydrolase I and II
Formation by Trichoderma reesei . Appl. Environ. Microb ., 57: 630-635.
Montenecourt, B.S and D.E. Eveleigh. 1977. Semi-quantitative plate assay for determination of
cellulase production by
Trichoderma viridii . Appl. Environ. Microb ., 33: 178-183.
Muthuvelayudham, R and T. Viruthagiri. 2006. Fermentative production and kinetics of cellulase
protein on Trichoderma reesei using sugarcane bagasse and wheat straw. Afr. J. Biotechnol .,
5: 1873-1888.
Nagendran, S., H.E. Hallen-Adams, J.M. Pape r, N. Aslam and J.D. Walton. 2009. Reduced
genomic potential for secreted plant cell-wall-degrading enzymes in the ectomycorrhizal fungus Amanita bisporigera , based on the secretome of Trichoderma reesei. Fungal Genet.
Biol., 427-435.
Nevalainen, H., P. Suominen and K. Taimisto. 1994. On the safety of Trichoderma reesei . J.
Biotechnol ., 37: 193-200.

NIGHAT ASLAM ET AL .,
2902
Rauscher, R., E. Wurleitner, C. Wacenovsky, N. Aro, A.R. Stricker, S. Zeilinger, C.P. Kubicek, M.
Pentilla and R.L. Mach. 2006. Transcriptional regulation of xyln 1, encoding xylanase 1, in
Hypocrea jecorena . Eukaryot Cell ., 3: 447-456.
Reese, E.T and A. Maguire. 1969. A surfactants as stimulants of enzyme production by
microorganisms. Appl. Microb ., 17: 242-5.
Shamala, T.R. and K.R. Sreekantiah. 1985. Pr oduction of cellulases and D-xylanase by some
selected fungal isolates. Enzyme Microb. Technol ., 8: 178-182.
Stricker, A.R., R.L. Mach, L.H. de Graaff. 2008. Regulation of transcription of cellulases- and
hemicellulases-encoding genes in Aspergillus niger and Hypocrea jecorina (Trichoderma
reesei ). Appl. Microbiol. Biotechnol ., 78: 211-220.
Sun, H., X. Ge, Z. Hao and M. Peng. 2010. Cellulase production by Trichoderma sp., on apple
pomace under solid state fermentation. Afr. J. Biotechnol ., 9: 163-166.
Suto, M and F. Tomita. 2001. Induction and catabolite repression mechanisms of cellulase in fungi.
J. Biosci. Bioeng ., 92: 305-311.
Szijarto, N., Z. Szengyel, G. Liden and K. Reczey. 2004. Dynamics of cellulase production by
glucose grown cultures of Trichoderma reesei Rut-C30 as a response to addition of cellulose.
Appl. Biochem. Biotechnol ., 115: 113-116.
Teeri, T., P. Lehtovaara, S. Kauppinen, J. Salovuori and J. Knowles. 1987. Gene ., 5: 43-52.
Vogel, H.J. 1956. A convenient growth medium for Neurospora (medium N). Microb. Genet. Bull .,
13: 42-43.
Xiao, Z., R. Storms and A. Tsang. 2005. Microplate-based carboxymethylcellulose assay for
endoglucanase activity. Anal. Biochem ., 176-178.

(Received for publication 4 January 2010)