Purification and some properties of a novel chitosanase from Bacillus subtilis KH1

One of at least two chitosanases secreted in the culture filtrate of Bacill us subtilis KH1 was purified by two sequential DEAE Sepharose CL-6B chromat ographies, followed by Sephacryl S-100 HR gel chromatography. The purified enzyme was homogenous as judged by SDS-PAGE. It showed an estimated molecul ar weight and pi of 28,000 and 8.3, respectively. The enzyme drastically re duced the viscosity of highly deacetylated chitosan substrates, with the su bsequent 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 hydrol ysis 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 de tected, 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 chitosana se derived from Bacillus pumilus BN262 and to the deduced amino terminal se quences of Bacillus subtilis 168 and Bacillus amyloliquefaciens UTK chitosa nases.