一株产琼胶酶细菌的分离、鉴定及其琼胶酶基本性质

Isolation,identification and characterization of an agaraseproducing marine bacterial strain Stenotrophomonas sp. NTa

【目的】分离海洋来源的琼胶酶产生菌,对其进行分类鉴定,并研究其所产琼胶酶的基本酶学性质,为琼胶酶的应用研究及开发利用奠定基础。【方法】通过以琼脂为唯一碳源的选择培养基分离产琼胶酶的菌株;利用16S rRNA基因序列分析、表型和生理生化特征对菌株进行鉴定;通过DNS-还原糖法测定琼胶酶活性;利用显色底物法测定琼胶酶的类型;对菌株所产琼胶酶粗酶的酶学性质进行初步研究。【结果】分离到一株产琼胶酶的菌株NTa,16S rRNA基因序列分析显示该菌株属于寡养单胞菌属(Stenotrophomonas sp.);该菌株主要产胞外琼胶酶,可分泌α-琼胶酶和β-琼胶酶;琼胶酶粗酶的最适反应温度和pH分别为40℃和7.0,并且琼胶酶在温度低于30℃,pH为7.0-9.0时稳定;Ca2+对琼胶酶粗酶具有促进作用,Ag+、Fe2+、Ba2+、Mn2+、Cu2+、Zn2+和Fe3+均可不同程度地抑制酶的活性;EDTA对琼胶酶粗酶活性具有抑制作用;琼胶酶粗酶对检测的抑制剂、去垢剂及变性剂有较好的抗性。【结论】海洋细菌Stenotrophomonas sp.NTa是一种新型的产琼胶酶菌株,可同时分泌α-琼胶酶和β-琼胶酶,具有潜在开发利用价值。

[ Objective] To identify and characterize a marine bacterial strain producing agarase. [ Methods] The agaraseproducing bacterium was isolated from coastal sediments in Xiamen using agar as the sole carbon source. The strain was identified by the analyses of 16S rRNA gene sequence, phenotype and biochemical reactions. Agarase activity was determined by dinitrosalieylie acid method, and the category of agarase was assayed using chromogenie substrate. At last, the characteristics of agarase were determined. [ Results] The results of the 16S rRNA phylogenetic, phenotypic and biochemical analyses showed that： the agar-degrading bacterium NTa belonged to the genus Stenotrophomonas sp.. The strain could produce extracellular agarases, including a-agarase and β-agarase. The optimum temperature and pH of strain NTa agarase were 40℃ and 7.0, respectively. The enzymatic activity was stable below 30℃. It also showed stability over a pH range between 7.0 and. 0. Ca2＋ could activate agarase activity, and Na ＋ , K ＋ and Mg2 ＋ had no significant influence. However, Ag＋, Ba2＋, Fe2＋, Mn2＋, Cu2＋, Zn2＋ and Fe3＋ inhibited the enzyme activity. The enzymatic activity of stain NTa agarase was inhibited by EDTA. The agarase had good resistance to some inhibitors, detergents and denaturant. [ Conclusion] Stenotrophomonas sp. NTa is a new type of agarase-producing strain, which can produce both ot-agarase and β-agarase and has potential applications in the production of agaro-oligosaceharide.