嗜热真菌木聚糖酶1YNA的表达和定点突变  

Characterization and Site-directed Mutagenesis of Xylanase 1YNA from Thermomyces lanuginosus DSM10635

作　　者：付正[1] 张华山[2] 曾小英[1] 熊海容[1] 

机构地区：[1]中南民族大学生命科学学院,武汉430073 [2]湖北工业大学发酵工程省部共建教育部重点实验室,武汉430068

出　　处：《湖北农业科学》2011年第7期1488-1493,共6页Hubei Agricultural Sciences

基　　金：国家"863"计划项目(2007AA100601);人事部留学人员科技活动择优资助基金优秀项目(2008-86)

摘　　要：为了进一步提高嗜热真菌(Thermomyces lanuginosus DSM10635)本已较耐热的木聚糖酶1YNA的热稳定性。利用重叠延伸PCR技术从嗜热真菌基因组DNA中扩增获得了木聚糖酶1YNA的基因xyl,将其插入到大肠杆菌表达载体pET-22b(+)中,构建了重组表达质粒pET-22b(+)-xyl。该质粒转化到表达宿主大肠杆菌BL21(DE3)后获得了能成功表达的工程菌。工程菌表达木聚糖酶1YNA的最适温度为65℃,最适pH值为6.0。其在75℃,pH值为6.5的条件下失活处理30min后还残存有30%的酶活,与嗜热真菌生产的木聚糖酶特性相近。在对该木聚糖酶的N端氨基酸序列进行定点突变后发现,T4A、T4G、T4R均对该木聚糖酶的热稳定性无显著影响,其中T4G突变体的耐热性有变弱的趋势。Overlap extension PCR technology was used to amplify xylanase 1YNA gene xyl from Thermomyces lanuginosus DSM10635.The PCR products were connected with expression vector pET-22b（＋） to construct recombinational vector pET-22b（＋）-xyl;and then transformed into expression host BL21（DE3）.The optimal expression condition of xylanase 1YNA was 65℃,pH 6.0.The xylanase remained 30% of activity after 30min inactivation at the condition of pH 6.5,75℃.A few N-terminus amino acids of xylanase 1YNA were changed by site-directed mutagenesis.The thermostability of xylanase mutants T4A and T4R was similar to their parent,while T4G showed weaker thermostability.

关 键 词：嗜热真菌 重叠延伸PCR 定点突变 木聚糖酶 

分 类 号：Q939.5[生物学—微生物学] Q78