杓兰果胶杆菌海藻糖酶的克隆表达及应用

Cloning, expression and properties of trehalase from Pectobacterium cypripedii

摘要:

海藻糖酶在工业发酵和食品医药等领域应用广泛，我国缺乏性能优良工业品种的海藻糖酶，同时在应用研究领域还不够深入。本研究从自然界筛选获得一株产酸性海藻糖酶的杓兰果胶杆菌(Pectobacterium cypripedii)，克隆获得其海藻糖酶基因PCTre，在大肠杆菌(Escherichia coli)BL21(DE3)体系中实现了重组表达，在5L发酵罐上28h产酶达到4130U/mL。酶学性质研究显示，PCTre能特异性水解海藻糖，最适反应温度和pH分别是35℃和5.5，在pH4.0、4.5、5.0处理8h后残余酶活仍超过80%，表现出良好的耐酸性。同时该酶还显示出优良的有机溶剂耐受性，在20%(V/V)乙醇溶液中处理24h仍能保留60%的酶活。进一步在含有20%(V/V)乙醇和7.5%海藻糖的模拟发酵体系中，当添加500U/L PCTre，可在16h内完全水解海藻糖，具有良好的应用于工业乙醇发酵的潜力。

关键词:海藻糖酶;海藻糖;杓兰果胶杆菌;乙醇耐受性

Abstract:

Trehalase is widely used in industrial fermentation, food, medicine and other fields. There is a lack of industrial varieties of trehalase with excellent performance in China. Moreover, the applied research on trehalase was not well conducted. In this study, a strain of Pectobacterium cypripedii was screened from nature, and the gene PCTre encoding an acidic trehalase was cloned and expressed in E.coli BL21(DE3). The highest enzyme activity reached 4130 U/mL after fermenting in a 5 L fermenter for 28 h. The enzymatic properties study showed that PCTre hydrolyzed trehalose specifically. The optimum pH and temperature were 5.5 and 35℃, respectively. 80% of the enzyme activity was retained after being treated at pH 4.0, 4.5, and 5.0 for 8 h, showing good acid tolerance. Moreover, it has good tolerance to organic solvents, 60% enzyme activity was retained after being treated with 20% (V/V) ethanol solution for 24 h. Furthermore, trehalose could be completely hydrolyzed within 16h in a simulated fermentation system containing 20% (V/V) ethanol and 7.5% trehalose, with 500 U/L PCTre added. This indicated a good application potential for industrial ethanol fermentation.

Key words:trehalase;trehalose;Pectobacterium cypripedii;ethanol tolerance