Partial Purification and Characterization of Cellulases by Bacillus alcalophilus S39 and B. amyloliquefaciens C23

Abstract

Maximum cellulase partial purification was achieved with 80 % (NH4)2SO4 precipitation. The Km and Vmax values were calculated for CMCase, FPase & β-glucosidase. It was found that Km values (0.92±0.28, 3.70, 0.55± 0.17 %) & (0.59±0.24, 3.86±0.39 & 0.65±0.14 %) and Vmax values (4.27± 0.73, 2.50 & 6.34±1.22 U/ ml) & (3.53±0.60, 2.22 & 7.42±1.04 U/ ml) for Bacillus alcalophilus S39 and B. amyloliquefaciens C23, respectively. It was found that the enzymes activity has a broad pH range between 4.8 and 5. The optimum temperature of the enzymes was observed to be around 50 °C, and 97 - 98 % of the original activity was retained after heat treatment at 80 °C for 15 min. The enzymes activity was stimulated by Ca++ & Co++, weak inhibition of cellulases with EDTA, Cu++, Acetone & Methanol, SDS & ethanol was the strongest inhibitor and the enzymes had no effect with Na+, Mn++, Tween 80 and Mg++. Analyses of the enzyme preparation by SDS-PAGE revealed two protein bands showing cellulolytic activity. The molecular weight of these bands was estimated to be around 66.49 and 28.47 KDa for Bacillus geneus.