海洋微生物几丁质酶分离纯化及其抗真菌活性

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

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Bsp1,经往复式摇床振荡培养96 h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23 ku,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;Zn2+、Cu2+和Hg2+能强烈抑制几丁质酶活性;Ni+和EDTA抑制20%～40%;然而5mmol/L Co2+可以使几丁质酶活性提高1.4倍;Mg2+、Ca2+等也能使酶活性增加。

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.