黑曲霉耐热木聚糖酶XYNB在毕赤酵母中的高效表达  

High Expression of Thermostable Xylanase XYNB from Aspergillus niger in Pichia pastoris

作　　者：李伟 何容[1] 高志强[1] 于荣清[1] 赵锐[1] 曹毅[1] 乔代蓉[1] 

机构地区：[1]四川大学生命科学学院微生物与代谢工程四川省重点实验室,成都610064

出　　处：《应用与环境生物学报》2011年第6期892-896,共5页Chinese Journal of Applied and Environmental Biology

基　　金：四川省科技支撑计划项目(No.2008GZ0245);科技人员服务企业行动项目(No.2009GJF00033)资助~~

摘　　要：高比活木聚糖酶的高效表达是进一步提高木聚糖酶发酵效价、降低生产成本的有效途径.将黑曲霉木聚糖酶基因XynB(不含信号肽)克隆到分泌型表达载体pPIC9K上,线性化后电击转化巴斯德毕赤酵母GS115,G418和PCR鉴定的阳性转化子经0.5%甲醇、在28℃诱导表达.SDS-PAGE分析表明,该蛋白相对分子质量为20×103左右.优化的诱导表达条件为,每隔12 h添加0.5%的甲醇,发酵5 d后,比活达4 757 U/mg;其最适温度为55℃,最适pH为5.0,80℃处理30min后仍有74%的残余酶活.The high expression of xylanase with high specific activity is the effective way to further improve the fermentation potency of xylanase and cut down the production costs. In this study, a recombinant plasmid pPIC9K-XynB was constructed by inserting gene XynB （without signal peptide） from Aspergillus niger into Pichia pastoris secretary vector pPIC9K. Linearized pPIC9K-XynB was transformed into P. pastoris GS115 by electroporation. The positive recombinant strain identified by G418 selection and confirmed by PCR analysis was induced by 0.5% methanol at 28 ℃ to express the recombinant xylanase. The SDS-PAGE analysis showed that the protein molecular weight was about 20 000. The enzyme production was high and stable （4 757 U/mg） when the methanol induced time interval was 12 h and the methanol concentration was 0.5%. The optimal temperature and pH of the enzyme activity was 55 ℃ and 5.0, respectively. After treated for 30 min at 80 ℃, there was still 74% residual activity. Fig 9, Ref 18

关 键 词：黑曲霉 木聚糖酶 毕赤酵母GS115 甲醇 诱导表达 

分 类 号：Q936[生物学—微生物学] Q786