Production, purification and characterization of a novel thermotolerant endoglucanase (CMCase) from Bacillus strain isolated from cow dung

Sangrila Sadhu, Pradipta Saha, Sukanta K Sen, Shanmugam Mayilraj, Tushar Kanti Maiti

In an attempt to screen out cellulase producing bacteria from herbivorous animal fecal matter it was possible to isolate a potent bacterium from cow dung. The bacterium was identified as Bacillus sp. using 16S rDNA based molecular phylogenetic approach. The effect of different agricultural wastes, paper wastes and carboxymethyl cellulose on endoglucanase production was tested and was found to produce maximally at 8% carboxymethyl cellulose. The endoglucanase was precipitated by ammonium sulfate saturation and purified by DEAE- Sepharose column. The purification was achieved 8.5 fold from the crude extract with a yield of 68.1%. The molecular weight of the protein was determined to be 97 kDa by SDS-PAGE. The enzymatic activity was moderately reduced by detergents (SDS, Tween-80), metal ions (MnCl2, ZnCl2) and EDTA. The endoglucanase was stable between pH 5.0 - 9.0 and temperature between 20-70°C with optimal activity at pH 7.0 and temperature 50°C. The apparent Km value of the enzyme for the substrate carboxymethyl cellulose was recorded to be 0.25 mg/ml. The endoglucanase was stable in the presence of commercial detergents such as Ariel, Surf Excel and Tide, indicated might be of potential applications in detergent industry. The enzyme from this strain could also be applied in bioconversion of lignocellulosic biomass into fermentable sugars.