固态发酵贵州绿僵菌产壳聚糖酶的亲和层析纯化及其性质研究

Purification of a Novel Chitosanase from Metarhizium guizhouense in Solid-state Fermentation with Affinity Chromatography

目的寻找壳聚糖酶新酶源并简化壳聚糖酶纯化工艺。方法将贵州绿僵菌固态发酵物粗提液经交联壳聚糖树脂层析,以5‰Cu2+洗脱,洗脱液透析浓缩后经Superdex75层析。结果该菌株在发酵120h时,每克干培养基酶活力可达(83.09±2.9)U。经亲和层析和分子筛分离可获得电泳纯的壳聚糖酶,酶活力回收率为43.52%。每克干培养基可获壳聚糖酶328μg,比活达106.39U/mg。酶蛋白相对分子质量为50.3×103。结论固态发酵贵州绿僵菌生产壳聚糖酶可作为壳聚糖酶的新酶源。用交联壳聚糖树脂亲和层析、Cu2+洗脱和分子筛分离的方法可以纯化壳聚糖酶。

Objectives To find a new source of chitosanase from Metarhizium guizhouense in solid state fermentation through purification techniques with affinity chromatography and molecular sieving. Methods Cross-linked chitosan resins adsorbed chitosanase from crude enzyme, which was then eluted with 5‰Cu^2＋. The elute was dialyzed, concentrated and separated by Superdex75 chromatography. Results Under the optimal conditions, the chitosanase activity reached a maximum of 83.09±2. 9 U for one gram of dry medium after 120 hours of culture. The chitosanase was purified, with 43.52% enzyme activity maintained. The specific activity of the purified enzyme reached 106.39 U/mg. The chitosanase had a molecular mass of 50.3×10^3. Conclusion A new source of chitosanase from Metarhizium guizhouense under solid state fermentation conditions is obtained. Affinity chromatography and molecular sieving can be used to purify chitosanase.