REACTION-MECHANISM OF CHITOSANASE FROM STREPTOMYCES SP N174

Chitosanase was produced by the strain of Streptomyces lividans TK24 b earing the can gene from Streptomyces sp. N174, and purified by S-Seph arose and Bio-Gel A column chromatography. Partially (25-35%) N-acetyl ated chitosan was digested by the purified chitosanase, and structures of the products were analysed by NMR spectroscopy. The chitosanase pr oduced hetero-oligosaccharides consisting of D-GlcN and GlcNAc in addi tion to glucosamine oligosaccharides [(GlcN)(n), n = 1, 2 and 3]. The reducing- and non-reducing-end residues of the hetero-oligosaccharide products were GlcNAc and GlcN respectively, indicating that the chitos anase can split the GlcNAc-GlcN linkage in addition to that of GlcN-Gl cN. Time-dependent H-1-NMR spectra showing hydrolysis of(GlcN)(6) by t he chitosanase were obtained in order to determine the anomeric form o f the reaction products. The chitosanase was found to produce only the alpha-form; therefore it is an inverting enzyme. Separation and quant ification of (GlcN)(n) was achieved by HPLC, and the time course of th e reaction catalysed by the chitosanase was studied using (GlcN)(n) (n = 4, 5 and 6) as the substrate. The chitosanase hydrolysed (GlcN)(6) in an endo-splitting manner producing (GlcN)(2), (GlcN)(3) and (GlcN)( 4), and did not catalyse transglycosylation. Product distribution was (GlcN)(3) much greater than (GlcN)(2) > (GlcN)(4). Cleavage to (GlcN)( 3) + (GlcN)(3) predominated over that to (GlcN)(2) + (GlcN)(4). Time c ourses showed a decrease in rate of substrate degradation from (GlcN)( 6) to (GlcN)(5) to (GlcN)(4). It is most likely that the substrate-bin ding cleft of the chitosanase can accommodate at least six GlcN residu es, and that the cleavage point is located at the midpoint of the bind ing cleft.