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.
Highlights
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
Screening Bacillus sphaericus CE-3 for carboxymethyl cellulase production was carried out by plate method [16]
Screening for carboxymethyl cellulase (CMCase) production showed a clear zone around the Bacillus sphaericus CE-3 organism, indicating a positive result
Summary
A polymer of β-1,4-linked glucose unit, is a major polysaccharide con-. Plants produce about 4 × 109 tons of cellulose annually along with other polysacharides [2]. Cellulose is an abundant and renewable energy source which can be converted to useful products (sugars, alcohols), and other industrially important chemicals by enzymatic degradation [3]. It has been reported that effective biological hydrolysis of cellulose into glucose requires synergistic actions of three enzymes, including endo-β-1,4-glucanase (EC3.2.1.4,EG, randomly cleaving internal linkages), cellobiohydrolase (EC3.2.1.91, CBH, hydrolyzing cellobiosyl units from non-reducing ends) and B-D-glucosidase (EC.3.2.1.21, hydrolyzing glucosyl units from cellooligosaccharides) [4]. Celluloses are becoming increasingly important and could provide key opportunity to achieving tremendous benefits in biomass utilization [1] and serve as substitutes for diminishing fossil energy resource [2]
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