Purification, characterization and cloning of a chitinase from Bacillus sp. NCTU2

A chitin‐degrading Bacillus strain, designated as NCTU2, was screened from soil and identified. An extracellular chitinase was purified to >90% homogeneity from the culture filtrate. The purification involved hydrophobic‐interaction and gel‐filtration chromatographic separations with a yield of 58%. The purified enzyme (ChiNCTU2) is a monomeric protein with an estimated molecular mass of 36·5 kDa and a pI of 6·3. It is thermally stable at 60 °C and pH 6–8 for more than 3 h. The optimal activity is in the range of 50–60 °C at pH 7·0. Chitobiose is the predominant product throughout the enzymic hydrolysis of the colloidal chitin, indicating that the purified chitinase is an exo‐chitinase. Chito‐oligosaccharides [with degree of polymerization (DP) values of 4–6] are good substrates of the purified enzyme, whereas a DP3 oligomer was slowly hydrolysed to form DP1 and DP2 sugars. The first 15 N‐terminal amino acids of the enzyme were determined to be ANNLGSKLLVGYWHN, which is highly homologous to that of ChiA from Bacillus cereus. A PCR cloning technique was employed to obtain the corresponding gene from Bacillus NCTU2. The gene sequence was determined to be 1080 bp, encoding a polypeptide of 360 amino acids with the first 27 amino acids as the signal peptide.