Clostridium clariflavum GH10木聚糖酶的克隆表达、酶学性质及位点功能分析

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  • 1(江南大学 工业生物技术教育部重点实验室,江苏 无锡,214122) 2(江南大学 生物工程学院,江苏 无锡,214122)

网络出版日期: 2018-07-31

基金资助

111引智计划(111-2-06);江苏省现代工业发酵协同创新中心资助

Cloning, expression, characterization and functional analysis of GH10 xylanase from Clostridium clariflavum

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  • 1(Key Laboratory of Industrial Biotechnology, Jiangnan University, Wuxi 214122, China) 2(School of Biotechnology, Jiangnan University, Wuxi 214122, China)

Online published: 2018-07-31

摘要

首次将来源于Clostridium clariflavum的木聚糖酶基因Clocl-2441中的糖苷水解酶家族10(glycosyl hydrolase families,GH)结构域基因连接到pET28a(+)载体上,在E.coli BL21(DE3)成功异源表达。经镍柱和脱盐分离纯化达到电泳纯,并对其酶学性质进行解析。结果表明,重组木聚糖酶rXyn2441GH10最适温度和pH分别为70 ℃和7.0,属于中性嗜热木聚糖酶。rXyn2441GH10在65 ℃以下和pH 4.0~9.0范围比较稳定,金属离子中5 mmol/L的Mg2+可以提高79.2%的酶活。以榉木木聚糖为底物重组酶的动力学参数,Vmax为1 691.5 μmol/(mg·min),Km值为2.5 mg/mL,kcat为1 236.4/s,kcat/Km为494.6 mL/(mg·s)。定点饱和突变表明209位点的组氨酸对酶活有重要的作用。

本文引用格式

王华广 , 刘雨露 , 胡方觊 , 等 . Clostridium clariflavum GH10木聚糖酶的克隆表达、酶学性质及位点功能分析[J]. 食品与发酵工业, 2018 , 44(6) : 16 -23 . DOI: 10.13995/j.cnki.11-1802/ts.016821

Abstract

The researchers are looking for new xylanase sources as well as molecular modification of existing xylanase. In this study, the recombinant pET28a (+) vector containing glycosyl hydrolase families 10 domain gene of xylanase Clocl-2441 from the Clostridium clariflavum was constructed and transformed to E. coli BL21 (DE3). The rXyn2441GH10 was expressed as soluble protein and purified by Ni2+-NTA affinity chromatography. The enzymatic properties analysis showed that the optimal temperature and pH value of recombinant xylanase rXyn2441GH10 were 70 ℃ and 7.0 respectively, which indicated that rXyn2441GH10 was a neutral thermophilic xylanase. rXyn2441GH10 was stable below 65 ℃ and at pH 4.0-9.0. 5 mmol/L Mg2+ increased the enzyme activity by 79.2%. The kinetic parameters of rXyn2441GH10 for beech xylan were as follows: Vmax was 1 691.5 μmol/(mg·min), Km was 2.5 mg/mL, kcat was 1 236.4/s, kcat/Km was 494.6 mL/(mg·s). Site-directed saturation mutations indicated that histidine at position 209 had an important role on enzyme activity.
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