Cloning and expression of a chitinase gene from Thermoactinomyces vulgaris KFB-C100

We have found that Thermoactinomyces vulgaris KFB-C100 produces a chitinase. The optimum temperature and pH of the enzyme activity were 55°C and 6.5. The enzyme was stable after heat treatment at 80°C for 30 min and stable in acidic and basic conditions (pH 6.0~11.0). The thermostable endo- chitinase from Thermoactinomyces vulgaris KFB-C100 was cloned into the plasmid pBR322 by using E. coli DH5α as a host strain. The positive clone carrying a recombinant plasmid (pKCHI23) with a 4.1-kb fragment containing the chitinase gene was found. The recombinant plasmid was analyzed to determine the essential region for chitinase activity and obtained a 2.3-kb fragment, which was subcloned into pTrc99A using the PstI and SalI sites to construct pTrc99A/pKCHI23-3. The resulting plasmid exerted high chitinase activity upon transformation of E. coli XL1-Blue cells. Chitinase was overproduced 14 times more in the clone cells than in the wild-type cells and the enzyme was purified to homogeneity. The purified enzyme showed the similar properties as the native chitinase from T. vulgaris in terms of molecular Weight and substrate specificity. The catalytic action of the cloned enzyme was an endo type, producing chitobiose as a major reaction product.