Clostridium clariflavum DSM 19732木聚糖酶Xyn2441的克隆表达及条件优化

Cloning,expression and optimization of xylanase Xyn2441 from Clostridium clariflavum DSM 19732

Clostridium clariflavum DSM19732是一种颇具应用价值的木质纤维素降解菌，尤其对半纤维素的降解能力非常高，国外学者已开始关注其中木质纤维素降解酶类的结构与功能，国内尚无相关报道。作者首次从复合菌系RXS基因组DNA中扩增出C．clariflavum DSM19732的一个表达双功能木聚糖酶（糖苷水解酶家族（glycosyl hydrolase family，GH）10和11的木聚糖酶）的基因Clocl-2441，并将其与载体pET28a（＋）连接，在大肠杆菌E．coli BL21（DE3）成功表达。重组菌最佳诱导表达条件为：添加诱导剂时的菌体量OD600值约为1．25、诱导剂IPTG浓度为0．5mmol／L、诱导时间7～8h及诱导温度25℃，此时胞内重组酶比酶活达到34．92U／mg。经镍柱初步分离纯化后比酶活达713．16U／mg，纯化倍数20．83倍，回收率27％。重组酶的最适反应温度和pH分别为70℃和6．5。

Clostridium clariflavum DSM 19732 is a potential lignocellulose degrading bacterium with especial high capacity to degrade hemicellulose. Recently, researchers abroad have paid an attention to its lignocellulose degrading enzymes. The gene of a novel bifunctional enzyme （Xyn2441） consisting of two xylanase catalytic domains （glycosyl hydrolase family 10 and 11 ） of C. clariflavum DSM 19732, was amplified from a microbial consortium RXS and inserted into plasmid vector pET28a （ ＋ ）. The gene was expressed in E. coli BL21 （DE3）. The activity of recombinant xylanase was 34.92 U/mg under the optimal inducing condition of OD600 1. 25 , IPTG 0.5 mmol/L and 7 to 8 hours at 25 ℃. After purification by Ni2＋ -NTA affinity chromatography, the activity of rXyn2441 was 713.16 U/mg, and the optimal temperature and pH were 70℃ and 6.5 respectively.