Thermostable chitosanase from Bacillus sp strain CK4: Cloning and expression of the gene and characterization of the enzyme

A thermostable chitosanase gene from the environmental isolate Bacillus sp, strain CK4, which was identified on the basis of phylogenetic analysis of the 16S rRNA gene sequence and phenotypic analysis, was cloned, and its com plete DNA sequence was determined. The thermostable chitosanase gene was co mposed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30-kDa enzyme. The deduced a mino acid sequence of the chitosanase from Bacillus sp. strain CK4 exhibits 76,6, 15,3, and 14.2% similarities to those from Bacillus subtilis, Bacill us ehemensis, and Bacillus circulans, respectively. C-terminal homology ana lysis shows that Bacillus sp. strain CK4 belongs to cluster III with B. sub tilis, The gene was similar in size to that of the mesophile B. subtilis bu t showed a higher preference for codons ending in G or C. The enzyme contai ns 2 additional cysteine residues at positions 49 and 211. The recombinant chitosanase has been purified to homogeneity by using only two steps with c olumn chromatography. The half-life of the enzyme was 90 min at 80 degrees C, which indicates its usefulness for industrial applications. The enzyme h ad a useful reactivity and a high specific activity for producing functiona l oligosaccharides as well, with trimers through hexamers as the major prod ucts.