海底沉积物中几丁质酶的筛选、分离及活性分析

Screening,Identification and Enzymatic Properties Studies of Chitinase from Submarine Sediments

目的：从海底沉积物中筛选得到一株几丁质酶活性较高的菌株,分离纯化菌株分泌的几丁质酶,并对其活性进行酶学分析。方法：以中国南海北部湾的沉积物为样本,结合透明圈法及DNS法筛选菌株。采用几丁质结合能力分析及多种层析方法分离纯化菌株分泌的几丁质酶,对其中一种几丁质酶进行酶学性质分析。结果：共筛选获得21株几丁质酶产生菌,其中分泌的几丁质酶活性最高的为蜡样芽孢杆菌B04（Bacillus cereus strain B04）。发现该菌共分泌6种含有几丁质结合域的蛋白。从蜡样芽孢杆菌B04发酵液中,分离纯化得到分子量为36 k Da的几丁质酶。研究发现,该酶是蜡样芽孢杆菌B04的一种主要的几丁质酶,其最适p H为4.0,最适反应温度为60℃,在p H 3.0~10.0范围内活性稳定,Co2＋对其活力有明显促进作用,Ag＋有显著的抑制作用。结论：为几丁质酶的工业化应用提供了基础。

Objective： The aim is to screen a strain with high chitinase activity from submarine sediments,to separate chitinase secreted by purified strain,and to analyze its activity by enzymatic methods. Methods：Sediments from Beibu Gulf of South China Sea were chosen as samples. Bacterial strains were screened using the clear zone and DNS method. Chitin binding assay and multiple chromatography methods were used to separate chitinase secreted by purified strain,and studied the enzymatic properties of the purified chitinase. Results： 21 strains of chitinase producers were obtained. Among them,chitinase secreted by Bacillus cereus strain B04 has the highest chitinase activity. A 36 k Da chitinase from the fermentative broth of B. cereus strain B04 was purified. Studies showed that the optimum p H and reaction temperature for the enzyme was p H 4. 0 and 60℃,respectively. In p H 3. 0 ~ 10. 0,it has stable activity. Its activity was significantly enhanced by Co2 ＋and inhibited by Ag＋. Conclusion： The basis for the industrial application of chitinase was provided.