果胶裂解酶、木聚糖酶及果胶裂解酶、木聚糖酶基因在毕赤酵母中的表达  

Expression of pectin lyase gene、xylanase gene、pectin lyase gene and Xylanase gene in Pichia pastoris

作　　者：高秋芳[1,2] 郭安平[1] 孔华[1] 邓伟科[1,2] 贺立卡[1] 

机构地区：[1]中国热带农业科学院热带生物技术研究所/农业部热带作物生物技术重点开放实验室,海南海口571101 [2]海南大学农学院,海南海口570228

出　　处：《广东农业科学》2010年第8期1-5,共5页Guangdong Agricultural Sciences

基　　金：国家"863"计划项目(2007AA021307);国家公益性行业科研专项(3-41)

摘　　要：运用RT-PCR技术,从绿色木霉(AS3.3711)mRNA中扩增出木聚糖酶基因xyn2,并与质粒pPIC9K相连,构建了表达载体pPIC9K-xyn2,转化毕赤酵母GS115,并且将pPIC9K-xyn2和pPIC9K-PelC(果胶裂解酶)同时转化毕赤酵母GS115,通过组氨酸营养缺陷型筛选,G418高拷贝菌株筛选,以及摇瓶诱导表达筛选,分别得到一株高表达木聚糖酶毕赤酵母菌株X5和一株同时高表达木聚糖酶和果胶裂解酶毕赤酵母菌株PX6。同时,将一株高表达果胶裂解酶毕赤酵母菌株P2保存。重组酶酶学性质分析表明,P2和PX6分泌果胶裂解酶,X5和PX6分泌木聚糖酶最适温度均为50℃;P2分泌果胶裂解酶最适pH5.4,最大酶活为14.2U/mL;X5分泌木聚糖酶最适pH6.0,最大酶活为5.8U/mL;PX6分泌果胶裂解酶最适pH5.4,最大酶活为12.6U/mL,分泌木聚糖酶最适pH6.0,最大酶活为4.2U/mL。The xylanase gene fragment xyn2 was successfully amplified from mRNA of Trichoderma viride （AS3.3711） by RT-PCR （reverse-transcription PCR assay） technique,and then ligased with pPIC9K vector to construct t methanol induced expression procedures sequentially,a strain named X5 which could high yield xylanase and another strain named PX6 which could high co-express xylanase and pectin lyase were obtained respectively.The recombinase enzymatic properties of the two strains X5 and PX6 and another strain P2 which was kept by our own laboratory were analyzed.The results indicated that the highest activity of pectin lyase secreted by P2,xylanase by X5,pectin lyase by PX6 and xylanase by PX6 was 14.2,5.8,12.6,4.2 U/mL respectively;The optimum pH and temperature of pectin lyase were pH 5.4 and 50℃,respectively;The optimum pH and temperature of xylanase were pH 6.0 and 50℃,respectively.

关 键 词：果胶裂解酶 木聚糖酶 表达 毕赤酵母 

分 类 号：Q78[生物学—分子生物学]