嗜热毛壳菌CT2纤维二糖水解酶Ⅰ在毕赤酵母中的高效表达  

Effective expression of Chaetomium thermophilum CT2 cellobiohydrolaseⅠin Pichia pastoris

作　　者：刘守安[1] 李多川[1] 张燕[1] 郭芳先[1] 俄世瑾[1] 

机构地区：[1]山东农业大学环境生物系,泰安271018

出　　处：《菌物学报》2006年第2期256-262,共7页Mycosystema

基　　金：国家863计划资助项目(2003AA241162);国家自然科学基金(30270013;30170013)

摘　　要：嗜热毛壳菌ChaetomiumthermophilumCT2可产生具有重要工业生产价值的纤维素酶类。RT-PCR扩增cbh1成熟蛋白的编码基因,利用基因重组的方法构建可在毕赤酵母分泌表达系统中表达纤维二糖水解酶的重组表达载体,并转化毕赤酵母得到重组子。在毕赤酵母醇氧化酶AOX1基因启动子的作用下,重组蛋白得到高效表达,小规模发酵量达1.42mg/mL。表达蛋白分泌到培养基中,分子量约80kD;以脱脂棉为底物测得酶活为21U/mL。表达蛋白在60℃稳定,70℃保温60分钟仍保持90%的酶活力,具有较高的热稳定性。Chaetomium thermophilum CT2 produces extracellular cellulase with industrial value. PCR product consisting of the CBH I coding region without its signal sequences was cloned into the yeast secretive plasmid pPIC9K, which was then transformed into Pichia pastoris GS 115. Highly efficient production of the cellobiohydrolase Ⅰ were achieved in P pastoris under the control of the AOXI promoter, and the expressing level was 1.42mg/mL according to small-scale cultures. Recombinant cellobiohydrolase I had a molecular mass of 80 kDa, and the activity was up to 21U/mL.The recombinant enzyme was thermostable at 60℃ and remained 90% of its original activity after 60min at 70℃. The high level of fully active recombinant cellobiohydrolase I got from P pastoris makes this expression system attractive for fermentor and industrial applications.

关 键 词：纤维素酶 整合表达 羧甲基纤维素 

分 类 号：Q786[生物学—分子生物学]