海洋微生物几丁质酶分离纯化及其抗真菌活性

以实验室筛选的海洋产几丁质酶短芽胞杆菌属（Bacillus brevis sp．）菌株Bspl，经往复式摇床振荡培养96h后，发酵液先后采取了75％的硫酸铵盐析、透析、几丁质亲和层析、SDS—PAGE等方法对几丁质粗酶液进行分离纯化和鉴定。几丁质亲和层析一步纯化后，经过SDS—PAGE电泳测定该酶的分子量为23ku，其比活力为86．65．纯化倍数为1．707、产率为32．1％。纯化的几丁质酶能抑制病原真菌的生长，对病原真菌的拮抗作用具有广谱性。同时研究了几丁质酶的稳定性，以胶态几丁质为底物，分离的几丁质酶在pH7．5，55．0℃左右具有最大酶活性；Zn^2＋、Cu^2＋和Hg^2＋能强烈抑制几丁质酶活性；Ni^＋和EDTA抑制20％-40％；然而5mmol／LCo^2＋可以使几丁质酶活性提高1．4倍；Mg^2＋、Ca^2＋等也能使酶活性增加。

Separation and Purification of a Chitinase from Marine Microbe and Determination of the Antifungal Activity

After 30 qC cultivation for 96 hours, the chitinase from a Bacillus brevis sp. named Bspl was extracted by 75% ammonium sulfate precipitation and then dialysed. The obtained ehitinase was purified by chitin.based affinity chromatography. The purified chitinase showed a single band with a molecular weight of 23 ku in gel after SDS-PAGE. The chitinase displayed a 86.65 specific activity, 1. 707 purified fold, and a 32. 1% yield. The crude ehitinase showed the inhibition to a wide-range of pathogenic fungi. The purified ehitinase has maximal activity at pH7.5 and 55.0 ℃ , but Zn^2＋ , Cu^＋ and Hg^2＋ inhibited the activity while 5 mmol/L Co^2＋ increased its activity.