Abstract
One of at least two chitosanases secreted in the culture filtrate of Bacillus subtilis KH1 was purified by two sequential DEAE Sepharose CL-6B chromatographies, followed by Sephacryl S-100 HR gel chromatography. The purified enzyme was homogenous as judged by SDS-PAGE. It showed an estimated molecular weight and pI of 28,000 and 8.3, respectively. The enzyme drastically reduced the viscosity of highly deacetylated chitosan substrates, with the subsequent formation of chitooligosaccharides [(GlcN)n, n=2–6]. No activity toward carboxymethylcellulose (CMC), chitobiose (GlcN)2, or chitotriose (GlcN)3 was detected. Separation and quantification of products of hydrolysis of 10% (w/v) solutions of chitooligosaccharides, (GlcN)n, n=2–6, by HPLC showed the splitting of (GlcN)n, n=4–6, in an endo-splitting manner. Oligomers comprising higher units than the starting substrate were also detected, indicating transglycosylation activity. The amino terminal sequence of this enzyme (A-G-L-N-K-D-Q-K-R-R) is identical to that of the chitosanase derived from Bacillus pumilus BN262 and to the deduced amino terminal sequences of Bacillus subtilis 168 and Bacillus amyloliquefaciens UTK chitosanases.
