Isolation of a cellulase producing Bacillus cereus from cow dung and determination of the kinetic properties of the crude enzyme

Abstract

The objective of the study was to isolate a thermostable alkaline cellulase producing bacterial strain from diverse natural sources such as goat excreta, cow dung, tropical soil, and organic matter. Opened hot cellulose agar plates were transferred on to the selective carboxy methyl cellulose (CMC) agar media and incubated for 2 days at 37 oC. The samples were cultured on Nutrient Agar in order to isolate cellulolytic bacteria. The bacteria isolated were screened for cellulolytic activity using serial dilution and pour-plate method after which they were characterised. The bacterial isolate with the highest CMC hydrolytic capacity which was obtained from cow dung was selected for further studies. Based on the morphological, biochemical and cultural analysis, the strain from cow dung was been identified as Bacillus sp. Pure culture of this bacterial strain was grown overnight, DNA was extracted and 16S rDNA was amplified by thermocycler using universal primers. When the amplified 16S rDNA PCR product was sequenced using automated sequencer and sequence similarity search was done for the 16S rDNA sequence using BLAST, the unknown organism was identified as Bacillus cereus with the GenBank accession no AF290555. Cellulase from Bacillus cereus showed zero order kinetics for 20 minutes. The optimum pH for the crude cellulase from Bacillus cereus was 9.0 at 45oC and the temperature optimum was 45oC at pH 9.0. Michaelis constant for the cellulase from this isolate to soluble cellulose was 38.60 g-1L and Vmax was 3.32 µmol mL-1 min-1 at pH 9.0 and at 45oC. Since this cellulase enzyme was stable for more than an hour at 40oC and at alkaline pH values, it could be a good candidate for industrial applications.