曲霉CJ22-326内切壳聚糖酶的分离纯化和性质

Purification and Properties of Endo-Chitosanase ChiB from Aspergillus sp.CJ22-326

使用75%乙醇沉淀、CM SepharoseFF离子交换色谱、SephacrylS 200凝胶过滤色谱和PhenylSepharoseCL 4B疏水色谱分离纯化技术,对曲霉CJ22 326发酵液中的内切壳聚糖酶进行分离纯化和性质研究.结果表明,纯化后的内切壳聚糖酶ChiB经SDS PAGE凝胶电泳鉴定为单带,其相对分子质量为27700.采用SephacrylS 200凝胶过滤色谱测定的相对分子质量为27800.ChiB作用最适温度为65℃,最适pH值为6.0,75℃保温1h还残留40%酶活.1mmol/LMn2+对ChiB有强烈激活作用,2mmol/LCu2+,Ag+,Hg2+,Cd2+,Fe3+有强烈抑制作用.ChiB作用的最适底物为脱乙酰度95%的胶体壳聚糖.

An endo-chitosanase ChiB from the culture supernatant of Aspergillus CJ 22-326 was purified to an apparent homogeneity through ethanol fraction, CM-Sepharose FF chromatography, Sephacryl S-200 and Phenyl Sepharose CL-4B chromatography. The molecular weight of ChiB was estimated as 27 700 by SDS-PAGE, and 27 800 by gel filtration respectively. The best ChiB activity existed at pH 6.0 and 60 ℃, and the activity still retained 40% ability at 75 ℃ for 60 min. The enzyme activity was increased about 2 fold by the addition of 1 mmol/L Mn^(2+). However ,2 mmol/L Cu^(2+),Ag^(+),Hg^(2+),Cd^(2+),Fe^(3+)strongly inhibited the enzyme.The best substrate was 95% deacetylated colloid chitosan.