Asian Research Thesis Index

Abstract

In the present study research was undertaken to clone an endo-1, 4-β-glucanase gene (bglC) of glycoside hydrolase family 5 from moderately thermophilic bacterial strain Bacillus licheniformis ATCC 14580. After genomic DNA isolation and PCR amplification, bglC was cloned into Escherichia coli DH5α cells by using pTZ57R/T vector. Screening of positive clones was done through colony PCR and restriction digestion analysis. Endo-1, 4-β-glucanase gene bglC (1.5 kb) was further expressed in E. coli BL21 (DE 3) strain by ligating it into pET-22b (+) expression vector. Purification of recombinant enzyme was done using ammonium sulphate precipitation followed by immobilized metal affinity chromatography (IMAC) and gel filtration. The enzyme was purified to 5.75 fold having enzyme activity of 7.9 U/ml/min and specific activity of 52.66 U/mg and the molecular weight was found to be 56 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. Characterization of recombinant endo-1, 4-β-glucanase enzyme showed that it was stable over a pH range of 4-7 and retained 100% of its activity at 60 ̊C. Substrate specificity of purified endo-1, 4-β- glucanase showed that enzyme was most active towards CMC. Line-weaver Burk plot revealed Km and Vmax values of 3.8 % and 8.38 U/ml/min respectively. pka1 and pka2 of active site ionizable groups were determined to be 4.2 and 7.1 respectively using Dixon plot. Thermodynamic parameters for hydrolysis of CMC were found to be Ea=36.32 kJ/mol, ∆H= 34.12 kJ/mol, ∆S=-6.4 kJ/mol and Q10=0.47. The activity of endo-1, 4-β- glucanase was increased in prescence of Co2+ and Mg2+ whereas Cu2+ and Hg2+ greatly reduced the enzyme activity. Bioinformatic analysis showed that B. licheniformis endo-1, 4-β-glucanase possess 72% identity with endoglucanase from Geobacillus stearothermophilus. The purified endo-1, 4-β-glucanase enzyme was further used for biostoning of denim. The biochemical properties of endo-1, 4-β-glucanase proved it a valuable candidate for use in laundry and textile industries and for utilizing cellulose in industrial bioethanol production.

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