Production and Characterization of Crude 1, 4 - β - endoglucanase by Pseudomonas aeruginosa Using Corn (Zea mays) Cobs and Pawpaw (Carica papaya) Fibres as Substrates

Abstract

Aims: Lignocellulose is one of the most abundant sources of organic material in the world and it is a polysaccharide which consisted of cellulose, hemicelluloses and lignin. Cellulases are group of hydrolytic enzymes capable of degrading cellulose to smaller sugar components such as glucose units. This study aimed to isolate, screen a suitable 1,4-β-endoglucanase producing bacteria using inexpensive lignocellulosic substrates and optimize conditions for its production as well as characterize the crude enzyme. Study Design: One-Factor-at-a-Time Methodology (OFAT) was used for culture medium optimization and characterization of crude 1,4-β-endoglucanase from Pseudomonas aeruginosa. Place and Duration of Study: Department of Microbiology, Faculty of Science, University of Port Harcourt, Nigeria, between April 2014 and October 2015. Methodology: Soil samples were collected from refuse dump site, cow dung composite soil and forest soil and screened for cellulase production on Bushnell Haas agar medium supplemented with 0.1% (w/v) carboxymethylcellulose (CMC) using 1.0% congo red dye. Isolates were identified using standard cultural, morphological and biochemical methods. Corncobs and pawpaw fibres were evaluated for the enzyme production and optimization of medium and culture conditions in submerged fermentation was investigated for maximum 1,4-β-endoglucanase production. The crude enzyme produced was characterized using standard physiochemical parameters. Results: Nine (9) out of 21 bacterial strains isolated from soil samples showed positive potential for endoglucanase secretion but only one (1) isolate (CDB4) showing largest clear zone was selected after screening and used for further study. This isolate was identified as Pseudomonas aeruginosa using morphological and biochemical characteristics. Result revealed that the organism was able to secrete the enzyme when corncobs and pawpaw fibres were separately used as carbon sources. The optimal pH and temperature for 1,4-β-endoglucanase production  using  corncob  (3%) was found to be 5.5 and 45°C after 3rd day of cultivation,  while maximum enzyme yield was at 45°C  in pawpaw fibre having pH 6.0  and substrate concentration of 4% after 3rd day of fermentation. The optimum temperature and pH for crude 1,4-β-endoglucanase  activity were at 40°C and 6.5. The crude enzyme was most stable at pH 6.5 and 40°C, uninfluenced across a pH range of 5.5-6.5 and retained over 75% activity at 80°C after 2 h incubation. About 80.84% activity was lost when enzyme was incubated at pH 7. The enzyme was strongly activated by Co2+ and only slightly by Fe2+ and EDTA, while Zn2+, Ca2+, Hg2+ and Mg2+ elicited significant inhibition of the crude enzyme activity with highest repression exhibited by Zn2+. Conclusion: Results of this study revealed the ability of Pseudomonas aeruginosa to efficiently utilize both corncobs and pawpaw fibres without requirement for expensive pretreatment normally given to lignocellulosic residues. Therefore, this bacterium and its intrinsic metabolic capacity for endoglucanase production using inexpensive substrates at low concentration as well as the high pH stability and thermostability presented by the crude enzyme makes it useful in industrial processes.