Research And Development On A Chitosanase And Preparation Of Chitooligosaccharides By Enzymatic Hydrolysis

A strain with high chitosanase activity was isolated from soil samples and identified as Aspergillus fumigatus. With the strain mutation and improvement, Aspergillus fumigatus Jxsd-97 was obtained.The optimal compositions of the medium for chitosanase production by Aspergillus fumigatus Jxsd-97 were as follows(%,w/v): chitosan 1.6, peptone 0.4, MgSO₄·7H₂O 0.04, KH₂PO₄ 0.04, KCl0.05, FeSO₄·7H₂O 0.001, the initial pH6.0. The optimal cultivation conditions for chitosanase production by Aspergillus fumigatus Jxsd-97 were 250mL flask containing 50mL medium, 0.6 mL of inoculation, 28℃ and shaking cultivation at 180rpm for 96 hours. The chitosanase activity of fermented broth could reach 9.35U/mL when Aspergillus fumigatus Jxsd-97 was incubated in flask under the optimal cultivition conditions. The chitosanase from Aspergillus fumigatus Jxsd-97 strain was extracted by ethanol precipitation(75%, v/v), and purified by dialysis (MW: 8000 14400) and ion-exchange chromatography (SP-Sepharose FF colum), the results in SDS-PAGE show a single band for the purified chitosanase and the molecular weight was estimated to be 25kD. The chitosanase was identified as endo-type chitosanase by the analysis of enzymatic hydrolysis of chitosan with Thin Layer Chromatograph. The optimal temperature and pH for hydrolysis of chitosan by the chitosanase were 63℃ and 5.8, respectively; The chitosanase activity was stable in the pH range of 4.8⁸.0 and the temperature under 45℃; Mn²⁺, enhanced the enzyme activity significantly, whereas Hg²⁺, Ag⁺ , Fe³⁺, Cd²⁺, Pb²⁺. The N-terminal amino acid sequence of chitosanase was determined to be YNLPNNLKQ, which provides useful information for further gene cloning of this enzyme.Endo-type chitosanase was used to produce chitooligosaccharides. The optimum conditions were pH 5.8, 3% substrate concentration, enzyme content of 5U/g chitosan, and 45℃ for 3 hours, respectively. The yield of chitooligosaccharides is 95.9%.The separation and purification of chitooligosaccharides was studied. Macroporous ion exchange resins D155 and JK204 were used to separate chitooligosaccharides and decolor.