Isolation and Characterization of Cellulase-producing Bacteria from Sugar Industry Waste
American Journal of BioScience
Volume 7, Issue 1, January 2019, Pages: 16-24
Received: Jan. 29, 2019;
Accepted: Mar. 8, 2019;
Published: Mar. 29, 2019
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Farjana Islam, Department of Biochemistry and Molecular Biology, Shahjalal University of Science and Technology, Sylhet, Bangladesh; Department of Biochemistry and Molecular Biology, University of Rajshahi, Rajshahi, Bangladesh
Narayan Roy, Department of Biochemistry and Molecular Biology, University of Rajshahi, Rajshahi, Bangladesh
Cellulases are inducible enzymes that are synthesized by a large number of microorganisms during their growth on cellulosic materials. This study focuses on the isolation and screening of cellulase-producing bacteria from sugar industry waste (molasses) and characterization by morphological and biochemical analysis. Further, purification of cellulase was carried by ammonium sulfate precipitation and followed by column chromatography and molecular weight determined by SDS-PAGE. The isolated bacterial strains were grown on carboxymethyl cellulose (CMC) agar plate at various optimum conditions like pH, temperature, incubation period, carbon and nitrogen sources and substrate concentration. Three isolated strains showed clear hydrolyzing zone on agar plates containing CMC agar after Congo-red staining were identified as cellulase-producing bacteria. Based on cultural, morphological, and biochemical characteristics, the isolated strains were identified as Paenibacillus sp., Aeromonas sp., and Bacillus sp. Among the isolated strains, Paenibacillus sp. showed the capability for highest cellulase production (0.89 µmol ml-1 min-1) at optimal pH 7.0 and 40°C temperature on 24 hour of the incubation period at 1% CMC substrate concentration and was selected for further cellulase purification. In the final step of cellulase purification, the specific activity, purification fold and recovery were 1720 U/mg, 9.74 and 35.6%, respectively. The molecular weight of the purified enzyme was determined 66.9 kDa and the enzyme showed a high specificity to CMC substrate. The bacterial strains present in molasses have the potential for cellulase production. Substrate specificity of the purified cellulase indicates it to be an endo-β-1, 4-glucanase. The cellulase produced from the selected strain may benefit for industrial application.
Isolation and Characterization of Cellulase-producing Bacteria from Sugar Industry Waste, American Journal of BioScience.
Vol. 7, No. 1,
2019, pp. 16-24.
Romeo, Tony (2008). Bacterial biofilms. Springer, 258–263, Berlin. ISBN 978-3-540-75418-3.
Potprommanee L, Wang XQ, Han YJ, Nyobe D, Peng YP, Huang Q, Liu JY, Liao YL, Chang KL (2017). Characterization of a thermophilic cellulase from Geobacillus sp. HTA426, an efficient cellulase-producer on alkali pretreated of lignocellulosic biomass. PLoS One. 13;12(4):e0175004.
Klemm, Dieter; Heublein, Brigitte; Fink, Hans-Peter; Bohn, Andreas (2005). Cellulose: Fascinating Biopolymer and Sustainable Raw Material. Angew. Chem. Int. Ed. 44: 3358–93.
Bhat MK (2000). Cellulases and related enzymes in biotechnology. Biotechnol Adv. 1: 355-383.
Shanmugapriya K, Saravana PS, Krishnapriya, Manoharan M, Mythili A, Joseph S (2012). Isolation, screening and partial purification of cellulose from cellulose producing bacteria. International Journal of Advanced Biotechnology and Research 3: 509-514.
Liang YL, Zhang Z, Wu M, Wu Y, Feng JX (2014). Isolation, screening, and identification of cellulolytic bacteria from natural reserves in the subtropical region of China and optimization of cellulase production by Paenibacillus terrae ME27-1. Biomed Res Int. 2014:512497. doi: 10.1155/2014/512497.
Perez J, Munoz-Dorado J, de la Rubia T, Martinez J (2002). Biodegradation and biological treatments of cellulose, hemicellulose and lignin: an overview. Int. Microbiol. 5: 53–63.
Gaur R, Tiwari S (2015). Isolation, production, purification and characterization of an organic-solvent-thermostable alkalophilic cellulase from Bacillus allismortis RG-07. BMC Biotechnol. 15:19. doi: 10.1186/s12896-015-0129-9.
Abdelnasser SSI, Ahmed IE (2007). Isolation and Identification of New Cellulases Producing Thermophilic Bacteria from an Egyptian Hot Spring and Some Properties of the Crude Enzyme. Australian Journal of Basic and Applied Sciences 1: 473-478.
Chandara SKR, Snishamol C, Prabhu NG (2005). Cellulase Production by Native Bacteria Using Water Hyacinth as Substrate under Solid State Fermentation: Malaysian Journal of Microbiology 1: 25-29.
Islam F, Roy N (2018). Screening, purification and characterization of cellulase from cellulase producing bacteria in molasses. BMC Res Notes. 11(1):445. doi: 10.1186/s13104-018-3558-4.
Cherry JR, Fidantsef AL (2003). Directed evolution of industrial enzymes: an update. Curr Opin Biotechnol. 14: 438-443.
Nakamura K, Kappamura K (1982). Isolation and identification of crystalline cellulose hydrolyzing bacterium and its enzymatic properties. J Ferment Technol 60: 343-348.
Doi RH (2008). Cellulase of mesophilic microbes: cellulosome and non– cellulosome producers: Annals of New York Academy of Sciences 1125: 267–279.
Rasul F, Afroz A, Rashid U, Mehmood S, Sughra K, Zeeshan N (2015). Screening and characterization of cellulase producing bacteria from soil and waste (molasses) of sugar industry. International Journal of Biosciences. http://dx.doi.org/10.12692/ijb/6.3.230-238
Yin LJ, Huang PS, Lin HH (2010). Isolation of cellulase producing Bacteria and Characterization of the Cellulase from the Isolated Bacterium Cellulomonas Sp. YJ5. J Agric Food Chem 58: 9833-7.
Ando T, Chambost JP, Kotoujansky A, Cattano J, Barras F (1984). Mutants of Erwiniachrysanthemi defective in secretion of pectinase and cellulase. J Bacteriol, 160: 1199-1203.
Azhar A. Hussain, Mohamed S. Abdel-Salam, Hoda H. Abo-Ghalia, Wafaa K. Hegazy, Safa S. Hafez (2017). Optimization and molecular identification of novel cellulose degrading bacteria isolated from Egyptian environment. Journal of Genetic Engineering and Biotechnology 15 (1): 77-85.
Apun K, Jong BC, Salleh MA (2000). Screening and isolation of a cellulolytic and amylolytic Bacillussp from pith waste. J Gen Appl Microbes 46: 263-267.
Buchanan RE, Gibbons NE (1974). Bergey’s of determinative bacteriology America: United States of America, 529-563.
Miller GL (1959). Use of Dinitrosalisylic acid reagent for determination of reducing sugars. Anal Chem. 31: 426-428.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951). Protein measurement with the Folinciocalteu’s reagent. J. Biol. Chem 193: 265-275.
Haung XP, Monk C (2004). Purification and characterization of a cellulase from a newly isolated thermophilic aerobic bacterium Caldibacillus cellulovorans gen. nov. sp. World journal of Microbiology and Biotechnology 20: 85-92.
Gopinath SM, Shareef I, Ashalatha, Ranjit S (2012). Isolation, Screening and Purification of Cellulase from Cellulase Producing Klebsiellavaricola RBER3 (KF036184.1). International Journal of Science and Research, 2319-7064.
Irfan M, Safdar A, Syed Q, Nadeem M (2012). Isolation and screening of cellulolytic bacteria from soil and optimization of cellulase production and activity. Turkish Journal of Biochemistry- Turk J Biochem. 37: 287-293
Chundakkadu, K. (1998). Production of bacterial cellulases by solid state fermentation of banana wastes. Bioresou Techniol 69: 231-239.
Nandimath AP, Kharat KR, Gupta SG, Kharat AS (2016). Optimization of cellulase production for Bacillus sp. and Pseudomonas sp. soil isolates. African Journal of Microbiology Research 10: 410-419.
Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS (2002). Microbial cellulose utilization: fundamentals & biotechnology. Microbiology and Molecular Biology Reviews 66: 506-577.
Verma V, Verma A, Kushwaha A (2012). Isolation & production of cellulase enzyme from bacteria isolated from agricultural fields in district Hardoi, Uttar Pradesh, India. Pelagia Research Library 3: 171-174.
Maki ML, Broere M, Leung KT, Qin W (2011). Characterization of some efficient cellulase producing bacteria isolated from paper mill sludges and organic fertilizers. Int J Biochem Mol Biol 2011;2(2):146-154
Coughlan MP, Mayer F (1992). The cellulose-decomposing bacteria and their enzyme systems. In The Prokaryotes vol. 1, eds. Balows, A., Truper, H. G., Dworkin, M., Harder, W. & Schleifer, K. H. pp. 460–516. New York: Springer-Verlag. ISBN 0-38797258-7.
Fu X, Liu P, Lin L, Hong Y, Huang X, Meng X, Liu Z (2010). A novel endoglucanase Cel9P) from a marine bacterium Paenibacillus sp. BME-14. Appl Biochem Biotechnol 160:1627-1636.
Wang CM, Shyu CL, Ho SP, Chiou SH (2008). Characterization of a novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39. Lett Appl. Microbiol. 47(1):46-53.
Lee YJ, Kim BK, Lee BH, Jo KI, Lee NK (2006). Purification and characterization of cellulase produced by Baciilusamyloliquefaciens DL-3 utilizing rice hull. College of Natural Resources and Life Science 840: 604-714.