Characterization of three chitosanase isozymes isolated from a commercial crude porcine pepsin preparation

Three chitosanases designated PSC-I, PSC-II, and PSC-III were purified from commercial pepsin preparation by sequentially applying pepstatin A-agarose affinity chromotography, DEAE-Sephacel ion-exchange chromatography, Mono Q column chromatography, and Mono P chromatofocusing. With respect to chitosan hydrolysis, the optimal pHs were 5.0, 5.0, and 4.0 for PSC-I, PSC-II, and PSC-III, respectively; optimal temperatures were 40, 40, and 30 °C; and the Km's were 5.2, 4.0, and 5.6 mg/mL. The molecular masses of the three isozymes were ∼40 kDa, as estimated by both gel filtration and SDS-PAGE, and the isoelectric points were 4.9, 4.6, and 4.4, respectively, as estimated by isoelectrofocusing electrophoresis. All three chitosanase isozymes showed activity toward chitosan polymer and N,N′,N″-triacetylchitotriose oligomer. Most effectively hydrolyzed were chitosan polymers that were 68-88% deacetylated.