高比活木聚糖酶XYN-W及其突变体在酵母中的高效表达  

High-efficiency Expression of Xylanase XYN-W with High Specific Activity in Pichia pastoris

作　　者：罗建杰[1] 王亚茹[1] 袁铁铮[1] 柏映国[1] 黄火清[1] 罗会颖[1] 姚斌[1] 范云六[2] 

机构地区：[1]中国农业科学院饲料研究所,农业部饲料生物技术重点开放实验室,北京100081 [2]中国农业科学院生物技术研究所,北京100081

出　　处：《中国农业科技导报》2010年第5期80-85,共6页Journal of Agricultural Science and Technology

基　　金：国家863计划项目(2007AA100601);国家肉鸡产业技术体系(nycytx-42-G2-05)资助

摘　　要：将来源于瘤胃真菌Neocallimastix frontalis的高比活木聚糖酶基因xyn-w整合到毕赤酵母表达载体pPIC9上,通过电击转化得到重组转化子。SDS-PAGE分析和表达产物的研究表明,木聚糖酶基因xyn-w得到了高效分泌表达,在5 L发酵罐中木聚糖酶蛋白表达量达到1 mg/mL,酶活性达到13 000 IU以上。XYN-W的C端57个氨基酸序列为连接序列和锚定区域,利用PCR方法将此段序列去除,得到截短的木聚糖酶序列xyn-m。用相同方法构建高效表达XYN-M蛋白的毕赤酵母工程菌株,表达产物经纯化后进行酶学性质测定,结果表明,其比活为19 856.6 IU/mg,Kcat值为4 433.8 s-1,与纯化的XYN-W蛋白(比活性13 795.3 IU/mg、Kcat值2 717.1 s-1)相比,分别提高了43.9%和63.2%。xyn-w gene encoding a xylanase with high specific activity from ruminal fungus Neocallimastix frontalis was inserted into pPIC9 with the yeast α-mating factor and transformed into Pichia pastoris by electroporation to obtain the recombinants.It was shown that the recombinant xylanase xyn-w was high efficiently expressed and secreted into the supernatant medium by SDS-PAGE and expression products analysis.In 5 liter fermentor,xylanase protein was expressed by the level of 1 mg/mL and its activity was more than 13 000 IU /mL.Using PCR method,a truncated xyn-m was obtained by deleting the 57 amino acids at the C-terminus of the xylanase XYN-W,which were binding domains and anchered domains.Similarly,the P.pastoris strains with high expression of xylanase XYN-M were constructed and the enzyme character of purified product was analyzed.The result showed that the specific activity and Kcat of XYN-M was 19 856.6 IU/mg and 4 433.8 s-1 respectively,increased 43.9% and 63.2% compared to those of XYN-W（13 795.3 IU/mg and 2 717.1 s-1）,respectively.

关 键 词：木聚糖酶 毕赤酵母 XYN-W 高效表达 比活性改良 

分 类 号：Q786[生物学—分子生物学]