ABB  Vol.2 No.3 , June 2011
Purification and characterization of the kinetic parameters of cellulase produced from wheat straw by Trichoderma viride under SSF and its detergent compatibility
ABSTRACT
This paper reports the purification and characterization of kinetic parameters of cellulase produced from Trichoderma viride under still culture solid state fermentation technique using cheap and an easily available agricultural waste material, wheat straw as growth supported substrate. Trichoderma viride was cultured in fermentation medium of wheat straw under some previously optimized growth conditions and maximum activity of 398±2.43U/mL obtained after stipulated fermentation time period. Cellulase was purified 2.33 fold with specific activity of 105U/mg in comparison to crude enzyme extract using ammonium sulfate precipitation, dialysis and Sephadex-G-100 column chromatography. The enzyme was shown to have a relative low molecular weight of 58kDa by sodium dodecyl sulphate poly-acrylamide gel electrophoresis. The purified enzyme displayed 6.5 and 55oC as an optimum pH and temperature respectively. Using carboxymethyl cellulose as substrate, the enzyme showed maximum activity (Vmax) of 148U/mL with its corresponding KM value of 68µM. Among activators/inhibitors SDS, EDTA, and Hg2+ showed inhibitory effect on purified cellulase whereas, the enzyme activated by Co2+ and Mn2+ at a concentration of 1mM. The purified cellulase was compatible with four local detergent brands with up to 20 days of shelf life at room temperature suggesting its potential as a detergent additive for improved washing therefore, it is concluded that it may be potentially useful for industrial purposes especially for detergent and laundry industry.

Cite this paper
nullIqbal, H. , Ahmed, I. , Zia, M. and Irfan, M. (2011) Purification and characterization of the kinetic parameters of cellulase produced from wheat straw by Trichoderma viride under SSF and its detergent compatibility. Advances in Bioscience and Biotechnology, 2, 149-156. doi: 10.4236/abb.2011.23024.
References
[1]   Shallom, D. and Shoham, Y. (2003) Microbial hemicellulases. Current Opinion in Microbiology, 6, 219-228. doi:10.1016/S1369-5274(03)00056-0

[2]   Kaur, J., et al. (2007) Purification and characterization of two endoglucanases from Melanocarpus sp. MTCC 3922. Bioresource Technology, 98, 74-81. doi:10.1016/j.biortech.2005.11.019

[3]   Thongekkaew, J., et al. (2008) An acidic and thermostable carboxymethyl cellulase from the yeast Cryptococcus sp. S-2: purification, characterization and improvement of its recombinant enzyme production by high cell-den- sity fermentation of Pichia pastoris. Protein Expression and Purification, 60, 140-146. doi:10.1016/j.pep.2008.03.021

[4]   Bhat, M.K. (2000) Cellulases and related enzymes in biotechnology. Biotechnology Advances, 18, 355-383. doi:10.1016/S0734-9750(00)00041-0

[5]   Parry, N.J., et al. (2001) Biochemical characterization and mechanism of action of a thermostable ?-glucosidase purified from Thermoascus aurantiacus. Biochemical Journal, 353, 117-127.

[6]   Kirk, O., Borchert, T.V. and Fuglsang, C.C. (2002) Industrial enzyme applications. Current Opinion in Biotechnology, 13, 345-351. doi:10.1016/S0958-1669(02)00328-2

[7]   Cherry, J.R. and Fidantsef, A.L. (2003) Directed evolution of industrial enzymes: an update. Current Opinion in Biotechnology, 14, 438-443. doi:10.1016/S0958-1669(03)00099-5

[8]   Pérez-Guerra, N., et al. (2003) Main characteristics and applications of solid-state fermentation. Electronic Journal of Environmental, Agricultural and Food Chemistry, 2, 343-350.

[9]   Viniegra-Gonzáles, G., et al. (2003) Advantages of fungal enzyme production in solid state over liquid fermentation system. Biochemical Engineering Journal, 13, 157-167. doi:10.1016/S1369-703X(02)00128-6

[10]   Xia, L. and Cen, P.L. (1999) Cellulase production by solid state fermentation on lignocellulosic waste from the xylose industry. Process Biochemistry, 34, 909-912. doi:10.1016/S0032-9592(99)00015-1

[11]   Gao, J., et al. (2008) Production and characterization of cellulolytic enzymes from the thermoacidophilic fungal Aspergillus terreus M11 under solid state cultivation of corn stover. Bioresource Technology, 99, 7623-7629. doi:10.1016/j.biortech.2008.02.005

[12]   Latifian, M., Hamidi-Esfahani, Z. and Barzegar, M. (2007) Evaluation of culture conditions for cellulose production by two Trichoderma reesei mutants under solid-state fermentation conditions. Bioresource Technology, 98, 3634-3637. doi:10.1016/j.biortech.2006.11.019

[13]   Leite, R.S.R., et al. (2008) Production and characteristics comparison of crude a-glucosidases produced by microorganisms Thermoascus aurantiacus and Aureobasidium pullulans in agriculture wastes. Enzyme and Microbial Technology, 43, 391-395. doi:10.1016/j.enzmictec.2008.07.006

[14]   Colombatto, D., Mould, F.L. and Bhat, M.K. (2003) Use of ?brolytic enzymes to improve the nutritive value of ruminant diets. A biochemical and in vitro rumen degradation assessment. Animal Feed Science and Technology, 107, 201-209. doi:10.1016/S0377-8401(03)00126-3

[15]   Dhillon, S.S., et al. (2004) Studies on the utilization of citrus peel for pectinase production using fungus Aspergillus niger. International Journal of Environmental Studies, 61, 199-210. doi:10.1080/0020723032000143346

[16]   Ahmed, I., Zia, M.A. and Iqbal, H.M.N. (2010) Bioprocessing of proximally analyzed wheat straw for enhanced production of cellulase through parameters optimization with Trichoderma viride under SSF. International Journal of Biological and Life Sciences, 6, 164-170.

[17]   Ghose, T.K. (1987) Measurement of cellulase activities. Pure and Applied Chemistry, 59, 257-268. doi:10.1351/pac198759020257

[18]   Lowry, O.H., et al. (1951) Protein measurement with folin phenol reagent. The Journal of Biological Chemistry, 193, 265-275.

[19]   De-Moraes, L.M.P., Filho, S.A. and Ulhaa, C.J. (1999) Purification and some properties of an alpha amylase and glucoamylase fusion protein from Saccharomyces cerevisiae. World Journal of Microbiology and Biotechnology, 15, 561-564. doi:10.1023/A:1008961015119

[20]   Sharma, J., et al. (2006) Partial purification of an alkaline protease from a new strain of Aspergillus oryzae AWT 20 and its enhanced stabilization in entrapped Ca-Alginate beads. http://www.ispub.com/ostia/index.php?xmlFilePath=journals/ijmb/vol2n2/awt.xml

[21]   Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of head of bacteriophage T4. Nature, 227, 680-685. doi:10.1038/227680a0

[22]   Steel, R., Torrie, J. and Dickey, D. (1997) Principles and procedure of statistics. McGraw Hill Book Co Inc., New York.

[23]   Ojumu, T.V., et al. (2003) Cellulase production by Aspergillus flavus linn isolate NSPR 101 fermented in sawdust, bagasse and corncob. African Journal of Biotechnology, 2, 150-152.

[24]   Raghavarao, K.S.M.S., Ranganathan, T.V. and Karanth, N.G. (2003) Some engineering aspects of solid state fermentation. Biochemical Engineering Journal, 13, 127-135. doi:10.1016/S1369-703X(02)00125-0

[25]   Zadrazil, F. and Punia, A.K. (1995) Studies on the effect of particle size on solid state fermentation of sugar cane bagasse into animal feed using white rot fungi. Bioresource Technology, 54, 85-97. doi:10.1016/0960-8524(95)00119-0

[26]   Mehta, V.J., Thumar, J.T. and Singh, S.P. (2006) Production of alkaline protease from an alkaliphilic actinomycete. Bioresource Technology, 97, 1650-1654. doi:10.1016/j.biortech.2005.07.023

[27]   Sen, S., Veeranki, V.D. and Mandal, B. (2009) Effect of physical parameters, carbon and nitrogen sources on the production of alkaline protease from a newly isolated Bacillus pseudofirmus SVB1. Annals of Microbiology, 59, 531-538. doi:10.1007/BF03175142

[28]   Odeniyi, O.A., Onilude, A.A. and Ayodele, M.A. (2009) Production characteristics and properties of cellulase/po- lygalacturonase by a Bacillus coagulans strain from a fermenting palm-fruit industrial residue. African Journal of Microbiology Research, 3, 407-417.

[29]   Ogawa, K. (1990) Fractionation and purification of cellulases from Trichoderma viride. Bulletin of the Faculty of Agriculture - Miyazaki University, 36, 271-280.

[30]   Olama, Z.A., Hamza, M.A., El-Sayed, M.M. and Abdel-Fattah, M. (1993) Purification properties and factors affecting the activity of Trichoderma viride cellulose. Food Chemistry, 47, 221-226. doi:10.1016/0308-8146(93)90153-7

[31]   Qin, Y., et al. (2008) Purification and characterization of recombinant endoglucanase of Trichoderma reesei expressed in Saccharomyces cerevisiae with higher glycosylation and stability. Protein Expression and Purification, 58, 162-167. doi:10.1016/j.pep.2007.09.004

[32]   Kim, C.H. (1995) Characterization and substrate specificity of an endo-beta-1,4-D-glucanase I (Avicelase I) from an extra cellular multienzyme complex of Bacillus circulans. Applied and Environmental Microbiology, 61, 959-965

[33]   Beg, Q.K. and Gupta, R. (2003) Puri?cation and characterization of an oxidation-stable, thiol-dependent serine alkaline protease from Bacillus mojavensis. Enzyme and Microbial Technology, 32, 294-304. doi:10.1016/S0141-0229(02)00293-4

[34]   Fadel, M. (2000) Production physiology of cellulases and β-glucosidase enzymes of Aspergillus niger grown under solid state fermentation conditions. Online Journal of Biological Sciences, 1, 401-411.

[35]   Haddar, A., et al. (2009) Two detergent stable alkaline serine-proteases from Bacillus mojavensis A21: puri?cation, characterization and potential application as a laundry detergent additive. Bioresource Technology, 100, 3366- 3373. doi:10.1016/j.biortech.2009.01.061

[36]   Saha, B.C. (2004) Production purification and properties of endoglucanase from a newly isolated strain of Mucor circinelloides. Process Biochemistry, 39, 1871-1876. doi:10.1016/j.procbio.2003.09.013

[37]   Ekperigin, M.M. (2007) Preliminary studies of cellulase production by Acinetobacter anitratus and Branhamella sp. African Journal of Biotechnology, 6, 28-33.

[38]   Bakare, M.K., et al. (2005) Purification and characterization of cellulase from the wild-type and two improved mutants of Pseudomonas fluorescens. African Journal of Biotechnology, 4, 898-904.

[39]   Cascalheira, J.F. and Queiroz, J.A. (1999) Kinetic study of the cellobiase activity of Trichoderma reesei cellulose complex at high substrate concentrations. Biotechnology Letters, 21, 651-655. doi:10.1023/A:1005525015777

[40]   Lucas, R., et al. (2001) Production, purification, and pro- perties of an endoglucanase produced by the hyphomycete Chalara (syn. Thielaviopsis) paradoxa CH32. Journal of Agriculture and Food Chemistry, 49, 79-85. doi:10.1021/jf000916p

 
 
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