Purification and properties of chitosanase from Bacillus circulans MH-K1.

A chitosanase was concentrated from the culture broth of Bacillus circulans MH-K1 and was purified to homogeneity by CM-cellulose and gel permeation chromatography. The enzyme has a molecular weight of about 30, 000, its Km is 0.63mg chitosan/ml and its pI is 9.2. The maximum velocity of chitosan degradation by the enzyme was obtained at 50°C when pH was maintained at 6.5. The enzyme was stable within the range of 0-40°C and pH 4.0-9.0. p-Chloromercuribenzoate and the metal ions of Cu2+ Hg2+, Ni2+, and Zn2+ inhibited the enzyme activity.The enzyme degraded chitosan, glycolchitosan and CM-chitosan, but β-1, 4-glucans such as chitin or its derivatives and CM-cellulose were not susceptible to the enzyme.The degree of deacetylation of chitosan significantly affected its susceptibility to the enzyme action. The most susceptible substrate was 80% deacetylated chitosan, and the substrates with less than 40% deacetylation were not affected by the enzyme. It is suggested that the presence of N-acetylglucosamine residues in the molecule of chitosan play an important role in the recognition of the substrate by the enzyme. The enzyme showed an endo-splitting type of activity, and the end product of chitosan degradation contained a mixture of the dimer and trimer of glucosamine. The smallest of the substrates was a tetramer of glucosamine.