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

作　　者：高涵 龚劲松 汪子凯 苏畅 许正宏[3] 史劲松 GAO Han;GONG Jinsong;WANG Zikai;SU Chang;XU Zhenghong;SHI Jinsong(Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China;School of Life Sciences and Health Engineering,Jiangnan University,Wuxi 214122,Jiangsu,China;National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing,School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China)

机构地区：[1]江南大学生物工程学院糖化学与生物技术教育部重点实验室,江苏无锡214122 [2]江南大学生命科学与健康工程学院,江苏无锡214122 [3]江南大学生物工程学院粮食发酵与食品生物制造国家工程研究中心,江苏无锡214122

出　　处：《生物工程学报》2022年第12期4658-4668,共11页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(21978116)。

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5 L发酵罐上28 h产酶达到4130 U/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pH 4.0、4.5、5.0处理8 h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500 U/L PCTre,可在16 h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。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 16 h 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.

关 键 词：海藻糖酶 海藻糖 杓兰果胶杆菌 乙醇耐受性 

分 类 号：TQ920.1[轻工技术与工程—发酵工程] Q78[生物学—分子生物学]