AiM  Vol.7 No.4 , April 2017
Partial Purification and Characterization of Cellulase Produced by Bacillus sphaericus CE-3
Abstract: Cellulase is an enzyme produced by fungi, bacteria, protozoa and termite, that hydrolyze cellulose. They are known for their diverse applications in industry and medicine. The aim of this study is to purify and investigate cellulolytic properties of cellulase enzyme produced by Bacillus sphaericus CE-3 isolated from refuse dump in Nnamdi Azikiwe University, Awka, Nigeria. Enzyme was produced by submerged fermentation at 30°C for 30 h. The enzyme was purified to homogeneity by dialysis in 4M sucrose solution, ion-exchange chromatography on Q-Sepharose FF and by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B. The relative molecular mass of the enzyme was estimated using SDS-Polyacrylamide gel electrophoresis. Effects of temperature, pH and metals on enzyme activity and stability and the relative rate of hydrolysis of various substrates were also studied. The Purification fold for the enzyme was 7.8, with 66.4 μ/mg specific activity protein and overall yield of 35.8. The relative molecular mass range of the enzyme was estimated between 22.3 kDa - 26.3 kDa. The enzyme was optimally active at pH 9.0 and 40°C, stable at pH 9.0 and unusually retained over 90% activity between 50°C - 100°C after 30 min incubation. It was strongly activated by Mn2+ but inhibited by Ba2+, Co2+, Hg2+, Pb2+, Cu2+, Sr2+, Fe2+, Ca2+ and Zn2+. The cellulase displayed high catalytic activity with untreated sawdust, followed by carboxymethyl cellulose, while sodium hydroxide treated sawdust was the least hydrolyzed. Since the enzyme is thermo-stable, alkalophilic and could utilize natural wastes like sawdust as substrate, it is obvious that it would be of great use in textile, starch processing and pulp and paper industries.
Cite this paper: Ekwealor, C. , Odibo, F. and Onwosi, C. (2017) Partial Purification and Characterization of Cellulase Produced by Bacillus sphaericus CE-3. Advances in Microbiology, 7, 293-303. doi: 10.4236/aim.2017.74024.

[1]   Wen, Z., Liao, W. and Chen, S. (2005) Production of Cellulase by Trichoderma reesei from Diary Manure. Bioresource Technology, 96, 491-499.

[2]   Yin, L.-J., Lin, H.-H. and Xiao, Z.-R. (2010) Purification and Characterization of a Cellulase from Bacillus subtilis YJ1. Journal of Marine Science and Technology, 18, 466-471.

[3]   Mandels, M. (1985) Application of Cellulases. Biochemistry Society, 13, 414-416.

[4]   Perez, J., Munoz-Dorado, J., de la Rubia, T. and Martinez, J. (2002) Biodegradation and Biological Treatments of Cellulose, Hemicellulose and Lignin: An Overview. International Microbiology, 5, 55-63.

[5]   Walsh, G.A., Murphy, R.A., Kileen, G.F., Headon, D.R. and Power, R.F. (1995) Technical Note: Detection and Quantification of Supplemental Fungus β-Glucanase Activity in Animal Feed. Journal of Animal Science, 73, 1074-1076.

[6]   Begiun, P. and Aubert, J.P. (1994) The Biological Degradation of Cellulose. FEMS Microbiology Review, 13, 22-58.

[7]   Liu, B., Yang, Q., Zhou, Q., Song, J., Chen, D. and Liu, H. (2004) Cloning and Expression of the Endo-β-Glucanase III cDNA Gene from Trichoderma viride AS3.3711. Acta Ecologica Sinica, 25, 127-132.

[8]   Celestino, K.R.S., Cunha, R.B. and Felix, C.R. (2006) Characterization of a β-Glucanase Produced by Rhizopus microspores var microsporus and Its Potential for Application in the Brewing Industry. BMC Biochemistry, 7, 23.

[9]   Singh, J. (2013) Production of Carboxymethyl Cellulase by Bacillus shaericus JS1 Strain in Low Cost Agriculture Waste Medium. Research Journal of Biotechnology, 8, 11-20.

[10]   Cavaco-Paulo, A. (1998) Mechanism of Cellulase Action in Textile Processes. Carbohydrate Polymer, 37, 272-277.

[11]   Ozaki, K. and Ito, S. (1991) Purification and Properties of an Acid Endo-β-1,4-Glucanase from Bacillus sp KSM-330. Journal of General Microbiology, 137, 41-48.

[12]   Han, S.J., Yoo, Y.J. and Kang, H.S. (1995) Characterization of a Bifunctional Cellulase and Its Structural Gene. Journal of Biological Chemistry, 270, 26012-26019.

[13]   Mawadza, C., Ralni, H., Zvauya, R. and Bo, M. (2000) Purification and Characterization of Cellulases Produced by Two Bacillus Strains. Journal of Biotechnology, 83, 177-187.

[14]   Araffin, A., Abdullah, N., UmiKalsom, M.S, Shirai, Y. and Hassan, M.A. (2006) Production and Characterization of Cellulase by Bacillus pumilus EB3. International Journal of Engineering and Technology, 3, 47-53.

[15]   Rawat, R. and Tewari, L. (2012) Purification and Characterization of an Acid Thermophilic Cellulase Enzyme Produced by Bacillus subtilis Strain LFS3. Extremophiles, 16, 637-644.

[16]   Teather, R.N. and Wood, P.J. (1982) Use of Congo Red-Polysaccharide Interactions in Enumeration and Characterization of Cellulolytic Bacteria from the Bovine Rumen. Applied Environmental Microbiology, 43, 777-780.

[17]   Miller, G.L. (1959) Use of the Dinitrosalicyclic Acid Reagent for the Determination of Reducing Sugars. Analytical Chemistry, 31, 426-428.

[18]   Bradford, M.M. (1976) A Rapid and Sensitive Method for the Determination of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry, 72, 248-54.

[19]   Chung, M.C.M. (1987) Polyacrylamide Gel Electrophoresis. In: Jeyaseelin, K., Chung, M.C.M. and Kon, O.L., Eds., Gene and Proteins: A Laboratory Manual of Selected Techniques in Molecular Biology, KSU Press, Paris, 9-11.

[20]   Mohamed, S.A.S., Magdi, A.M.Y., Francis, F.H. and Moustafa, A.N. (2010) Production of Cellulase in Low Cost Medium by Bacillus subtilis KO Strain. World Applied Sciences Journal, 8, 35-42.

[21]   Padilha, I.Q.M., Carvalho, L.C.T., Dias, P.V.S., Grisi, T.C.S.L., Howataro da Silva, F.L., Santos, S.F.M. and Araujo, D.A.M. (2015) Production and Characterization of Thermophilic Carboxymethyl Cellulase Synthesized by Bacillus sp. Growing on Sugarcane Baggage in Submerged Fermentation. Brazilian Journal of Chemical Engineering, 32, 35-42.

[22]   Yoshimatsu, T., Ozaki, K., Shikata, S., Ohta, Y., Koike, K., Kawai, S. and Ito, S. (1990) Purification and Characterization of Alkaline Endo1-4-β-Glucanase from Alkalophilic Bacillus sp KSM-635. Journal of General Microbiology, 136, 1973-1979.

[23]   Singh, V.K. and Kumar, A. (1988) Production and Purification of an Extracellular Cellulase from Bacillus brevis VS1. Biochemistry and Molecular Biology International, 45, 443-452.

[24]   Yan, H., Dai, Y., Zhang, Y., Yan, L. and Liu, D. (2011) Purification and Characterization of an Endo-1,4-β-Glucanase from Bacillus cereus. African Journal of Biotechnology, 10, 16277-16285.

[25]   Aftab, S., Aftab, M.N., Ikram-UI-Haq, Javed, M.M. and Zafer, I. (2012) Cloning and Expression of Endo-1,4-β-Glucanase Gene from Bacillus licheniformis ATCC 14580 into Escherichia coli BL21 (DE 3). African Journal of Biotechnology, 11, 2846-2854.

[26]   Chan, K.Y. and Au, S.K.S. (1987) Purification and Properties of Endo 1,4-β-Glucanase from Bacillus subtilis. Journal of General Microbiology, 133, 2155-2162.

[27]   Uziie, M. and Sasaki, T. (1987) Purification and Properties of Cellulase Enzyme from Rhobillarda sp. Enzyme Microbiology and Technology, 9, 459-465.

[28]   Khyami-Horani, H. (1996) Partial Purification and Some Properties of an Alkaline Cellulase from an Alkalophilic Bacillus sp. World Journal of Microbiology and Biotechnology, 12, 525-529.