碱性木聚糖酶产生菌的筛选、XynG1-3基因克隆表达及酶学性质研究  

Screening of Alkaline Xylanase Producing Strains and Cloning,Expression and Characterization of Xylanase Gene XynG1-3

作　　者：郑宏臣[1] 刘逸寒[1] 刘晓光[1] 韩杨[1] 王建玲[1] 路福平[1] 

机构地区：[1]天津科技大学生物工程学院、工业发酵微生物教育部重点实验室、工业酶国家工程实验室、天津市工业微生物重点实验室,天津300457

出　　处：《生物技术通报》2012年第10期106-113,共8页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31101219)

摘　　要：从造纸厂周边碱性土壤中分离、筛选到一株产碱性木聚糖酶的细菌G1-3,根据形态和生理、生化特性并结合16SrDNA序列分析,鉴定菌株G1-3为短小芽孢杆菌,命名为Bacillus pumilusG1-3。利用克隆表达载体pET-22b(+),实现了Bacillus pumilusG1-3碱性木聚糖酶基因XynG1-3在E.coliBL21中的表达。经氨基酸序列分析,木聚糖酶XynG1-3属于GH11家族的小分子木聚糖酶。重组木聚糖酶XynG1-3经镍离子亲和层析一步纯化后,获得凝胶电泳条带单一的蛋白样品,经SDS-PAGE检测其分子量为24 kD。对酶学性质进行分析,重组酶XynG1-3最适作用温度为55℃,最适作用pH为8.0;该酶在60℃保温1 h,残余酶活保持为原来的56%,pH作用范围较广,在pH10.0下保存1 h,残余酶活仍能保持75%,为耐碱性木聚糖酶。A new alkaline xylanase-producing strain was isolated from alkaline soil around paper mill and was identified as Bacillus pumilus G1-3 based on the evidence from its physiological and biochemical characteristics and 16S rDNA sequences.The gene coding for XynG1-3 consists of 687 bp was cloned into the expression vector pET-22b and expressed in E.coli BL21（DE3）.XynG1-3 was determined to be a new xylanase belonging to glycoside hydrolase family 11 based on its amino acid sequence.The recombinant xylanase was purified by Ni2＋-NTA affinity chromatography.The molecular weight of the purified XynG1-3 was estimated to be 24 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.It revealed optimal activity at 55℃ and pH8.0 and remained high activity over a large range of pH（6.0-10.0）which remained at least 75% residual activity at pH 10.0 for 1 h.

关 键 词：短小芽孢杆菌 碱性木聚糖酶 筛选 鉴定 克隆 表达 酶学性质 

分 类 号：TQ925[轻工技术与工程—发酵工程]