Purification, characterization, and gene cloning of a chitosanase from Bacillus species strain S65

For the production of oligosaccharides from chitosan, a chitosanase-producing bacterium, S65, was isolated from soil. On the basis of phylogenetic analysis of the 16S rDNA gene sequence and phenotypic analysis, S65 was identified as a Bacillus sp. strain. This bacterium constitutively produced chitosanase in a culture medium without chitosan as an inducer. S65 chitosanase was homogeneously purified by DEAE Sepharose fast flow anion exchange followed by Superdex 75 size exclusion, and the molecular weight was 45 kDa according to SDS-PAGE. Enzyme analysis showed that the optimum pH and temperature of S65 were 6.0 and 65 degrees C, respectively. Catalytic activity was stable from pH 5.5-6.5 at temperatures below 40 degrees C, and the pl of chitosanase was about 6.0 as determined by a test tube method. S65 chitosanase degraded carboxymethyl cellulose (CMC) at the degree of about 5.3% relative to the value of soluble chitosan, but it cannot hydrolyze colloidal chitin and crystalline cellulose. Gene encoding was cloned and sequenced. The deduced amino acid sequence of the S65 exhibited the highest homology to those of family 8 glycanase, suggesting that the enzyme belonged to family 8.