Purification and characterization of two types of chitosanase from Aspergillus sp. CJ22-326

Aspergillus CJ22-326, a fungi strain capable of utilizing chitosan as a carbon source, was isolated from soil samples. Two types of chitosanase (ChiA and ChiB) produced from the culture supernatant of Aspergillus CJ22-326 were purified to an apparent homogeneity identified by SDS–PAGE through ammonium sulfate precipitation, CM-Sepharose FF chromatography, and Sephacryl S-200 gel filtration. Molecular weights of the enzymes were 109 kDa (ChiA) and 29 kDa (ChiB). Optimum pH values and temperature of ChiA were 4.0 and 50 °C, respectively, those of ChiB were 6.0 and 65 °C. The enzyme activities of ChiA and ChiB were increased by about 0.5-fold and 1.5-fold, respectively, by the addition of 1 mM Mn²⁺. However, 2.5 mM Ag⁺, Hg²⁺ and Fe³⁺strongly inhibited ChiA and ChiB activities. Viscosimetric assay and analysis of reaction products of these enzymes, using chitosan as a substrate, by TLC indicated endo- and exo-type cleavage of chitosan by ChiB and ChiA, respectively. ChiB catalysed the hydrolysis of glucosamine (GlcN) oligomers larger than pentamer, and chitosan with a low degree of acetylation (0–30%), and formed chitotriose with chitohexaose as the major products. ChiA released a single glucosamine residue from chitosan and glucosamine oligomers. Both of the activities of ChiA and ChiB increased with the degree of deacetylation of chitosan. The enzyme ChiB had a useful reactivity and a high specific activity for producing functional chitooligosaccharides with high degree of polymerization.