Purification and Characterization of Endoglucanase From Bacillus Licheniformis NA8

Abstract

Carboxymethyl cellulase(CMCase) activityin the the cell free supernatant of cellulose degrading bacteria Bacillus licheniformis NA8 was studied using 2% carboxymethyl cellulose. The protein was precipitated upto 60-80% saturation with ammonium sulphate and further purified using DEAE-Sepharose column. 4.76 fold purification of endoglucanase was achieved from crude extract with a yield of 83.49% through DEAE sepharose column chromatography. The molecular weight of the protein was 24 kDaas determined by SDS-PAGE and further confirmed by zymogram analysis. The endoglucanase was found to be stable between pH 5–7 and temperature between 30−60°C with optimal activity at pH 6.0 and temperature 50°C. The Km value of the enzyme for the substrate carboxymethyl cellulose was recorded to be 0.25 mg/mL. The purified cellulase was activated by Mn2+ and Co2+, but inhibited by Hg2+, Cd2+, Pb2+. The purified endo-1,4-glucanase showed maximum (2.01 ± 0.03 U/mL) substrate specificity for Carboxymethyl cellulose (CMC) followed by Avicel and filterpaper. The high catalytic activity and its stability to temperature, pH, surfactants, and metal ions indicated that the cellulase enzyme from B. licheniformis NA8 is good candidate for hydrolysis of lignocellulosic waste and biorefinery processes.