Application of response surface methodology to the modeling of cellulase purification by solvent extraction


Central composite design (CCD)sp. JS14 in a solvent extraction was established with Response surface methodology (RSM). Solvent concentration, pH, temperature and retention time were selected as process variables to evaluate the purification impact factor in solvent precipitation, including the purification fold and % recovery. An experimental space with 13 purification fold and 23 recovery percentage recovery is achieved through the optimized condition based on the model. The molecular weight of the purified enzyme was estimated to be 32.5 KDa. Optimum activity of purified enzyme was at pH and temperature 6.5℃ and 40℃ respectively. Enzyme showed maximum activity with carboxymethyl cellulose as substrate with compare to rice husk, wheat straw and sucrose. The purified cellulase activity was inhibited by Na+, Cl- Mg2+ Tween 80 and EDTA.

Share and Cite:

Singh, J. and Sharma, A. (2012) Application of response surface methodology to the modeling of cellulase purification by solvent extraction. Advances in Bioscience and Biotechnology, 3, 408-416. doi: 10.4236/abb.2012.34058.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Whitaker, J. (1990). Cellulase Production and Applications. Food Biotechnol, 4, 669-697.
[2] Solomon, B., Amigun, B., Betiku, E., Ojumu, T. and Layokun, S. (1997) Optimization of cellulase production by Aspergillus flavus Linn isolate NSPRI101 grown on bagasse. J. Nig. Soc. Chem. Eng, 16, 61-68.
[3] Beguin, P. and Aubert, J. (1994) The biological degration of cellulose. FEMS Microbiol. Rev, 13, 25-28.
[4] Kaur, J., Chadha, B., Kumar, A. and Saini, H. (2007) Purification and characterization of two endoglucanases from Melanocarpus sp. MTCC 3922. Bioresour. Technol, 98,74-81.
[5] Adsul, M., Bastawde, K., Varma, A. and Gokhale, D. (2007) Strain improvement of Penicillium janthinellum NCIM 1171 for increased cellulase production. Biores.Technol, 98, 1467-1473.
[6] Silva, C. and Roberto, C. (2001) Optimization of xylitol production by Candida guilliermondii FTI 20037 using response surface methodology. Process. Biochem, 36, 1119–1124.
[7] Bhat, M . (2000) Cellulases and related enzymes in biotechnology. Biotechnol. Adv, 18,355-383.
[8] Duff, S. and Murray, D. (1996) Bioconversion of forest products industry waste cellulosics to fuel ethanol: a review. Biores. Technol, 55, 1-33.
[9] Nieves, R., Ehrman, C., Adney, W., Elander, R. and Himmel, M. (1998) Technical communication: survey and analysis of commercial cellulase preparations suitable for biomass conversion to ethanol. W. J. Microb. Biotechnol, 14, 301-304.
[10] Chahal, S., Chahal, D. and Andre, G. (1992) Cellulase production profile of Trichoderma reesei on different cellulosic substrates at various pH levels. J. Ferment. Bioeng, 74,126-128.
[11] Reczey, K., Szengyel, R. and Zacchi,G. (1996) Cellulase production by T. reesei. Biores. Technol, 57, 25-30
[12] Dey, R., Mitra, M. and Banerjee, B. (2001) Enhanced production of amylase by optimization of nutritional constituents using response surface methodology. Biochem. Eng J. 227–231.
[13] Kristo, E., Biliaderis, C. and Tzanetakis, N. (2003) Modelling of the acidification process and rheological properties of milk fermented with a yogurt starter culture using response surface methodology. Food Chem, 83, 437–446.
[14] Chen, F., Cai, T., Zhao, G., Liao, X., Guo, L. and Hu, X. (2005) Optimizing conditions for the purification of crude octacosanol extract from rice bran wax by molecular distillation analyzed using response surface methodology. J. Food. Eng, 70, 47–53.
[15] Miller, G. (1959) Use of dinitrosalicylic acid regent for determination of reducing sugar. Anal. Chem, 31, 426-428.
[16] Lowry ,O., Rosebrough, N., Farr, V. and Randall, R. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem,193, 265–275.
[17] Laemmli, U. (1970) Cleavage of structural proteins during the assembly of the head of becteriophage T4. Nature (London), 227,680–5.
[18] Lineweaver, H. and Burk, D. (1934). The determination of enzyme dissociation constants. J. Amer. Chem. Soc, 56, 658-66..
[19] Eriksen, J. and Goksoyr, J. (1977) Cellulases from Chaetomium thermophile var. dissitum. Eur. J. Biochem,77,445–50.
[20] Ganju, R., Murthy, S. and Vithayathil, J. (1990) Purification and functional characteristics of an cellulase from Chaetomium thermophile var. coprophile. Carbohy. Res. 197:245–55.
[21] Hayashida, S. and Yoshioka, H. (1980) The role of carbohydrate moiety on thermostability of cellulases from Humicola insolens YH-8. Agric. Biol. Chem,44,481–487.
[22] Rao, U. and Murthy, S (1988) Purification and characterization of a glucosidase and cellulase from Humicola insolens. Indian J. Biochem. Biophysiol. 25:687–94.
[23] Roy K, Dey S, Raha S, Chakrabarty S(1990) Purification and properties of an extracellular endoglucanase from Myceliophthora thermophila D-14 (ATCC 48104). J. General Microbiol.136:1967–71.
[24] Sharma S, Malik. A. and Satya, S. (2009) Application of response surface methodology (RSM) for optimization of nutrient supplementation for Cr (VI) removal by Aspergillus lentulus AML05. J. Hazard. Mater, 164, 1198-1204.
[25] Gavin, M. and Forsberg, C. W. (1988) Isolation and characterization of endoglucanases 1 and 2 from Bacteroides succinogenes S85. J Bacteriol, 170(7), 2914–2922
[26] Tong, C., Cole, A. and Shephert, M. (1980) Purification and properties of the cellulases from the thermophilic fungus Thermoascus aurantiacus. Biochem. J, 191,83–94.
[27] Wood, T. M. and McCrae, S. I (1978) The cellulase of Trichoderma koningii. Purification and properties of some endoglucanase components with special reference to their action on cellulose when acting alone and in synergism with the cellobiohydrolase. Biochem J, 171(1), 61–72.

Copyright © 2023 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.