CBHⅡ和EGⅣ的重构基因在里氏木霉中的组成型表达  

Construction of CBHⅡ and EGⅣ for Homologous Protein Expression with Constitutive Promoter in Trichoderma Reesei

作　　者：曾敏 邓丽瑜[1] 汤新[1] 刘刚[1] 余少文[1] 

机构地区：[1]深圳大学生命科学学院,深圳市微生物基因工程重点实验室,广东深圳518060 [2]深圳大学生命科学学院,深圳市海洋生物资源与生态环境重点实验室,广东深圳518060

出　　处：《食品研究与开发》2015年第13期114-117,138,共5页Food Research and Development

基　　金：深圳市基础研究计划(JCYJ20120613115323982)

摘　　要：重构基因cbh2-linker-CDeg4表达的融合蛋白同时获得具有外切葡聚糖酶CBH II和内切葡聚糖酶EG IV 2种催化活性,在纤维素降解等方面有着重要应用。本研究通过overlap PCR将里氏木霉丙酮酸脱羧酶(PDC)的启动子、纤维二糖水解酶cbh1的信号肽、重构基因cbh2-linker-CDeg4和pdc终止子依次连接,以质粒p PICZαA为基本骨架,构建表达载体p PIC-PCT。采用原生质体转化法将p PIC-PCT和含潮霉素抗性筛选标记的质粒p AN7-1共转里氏木霉。SDS-PAGE分析表明,重构基因cbh2-linker-CDeg4实现了在里氏木霉中的组成型表达,测得重组木霉发酵上清液的CMCNa酶活最高达到4.08 U/m L,是出发菌株的5.55倍,FPA酶活达到0.915 U/m L,较出发菌株提高了43.6%。酶学性质初步研究表明:粗酶液的最适p H为5.0,最适温度为50℃。The reconstructed gene cbh2-linker-CDeg4 gain two kinds of cellulase catalytic properties ,which added the catalytic domain （CD） gene from eg4 to the 3＇ end of cbh2. The gene cbh2-linker-CDeg4 was inserted between T.reesei QM9414 strong promoter PSpdc （including secreting signal peptide sequence ） and terminator Tpdc, generating reconstructed plasmid pPIC-PCT. The plasmid pPIC-PCT and plasmid pAN7-1 were transformed into T.reesei via an improved protoplast transformation. SDS-PAGE analysis showed that the fusion protein CBHⅡ-EGⅣhad successfully expressed in T.reesei. The CMCNa activity of the T.reesei recombinants could reach 4.08 U/mL, which is 5.55-fold as high as that of the host strain; the FPA cativity could reach 0.915 U/mL, which were increased by 43.6%. The cellulase characterization of recombinant showed that the optimum pH value was about 5.0,and the optimum reaction temperature was at 50℃.

关 键 词：基因重构 外切葡聚糖酶Ⅱ 内切葡聚糖酶Ⅳ 丙酮酸脱羧酶启动子 

分 类 号：Q78[生物学—分子生物学]