内切葡聚糖酶基因cel7b在里氏木霉中的同源表达

Homologous expression of endoglucanase gene cel7b in Trichoderma reesei

用里氏木霉(Trichoderma reesei)作为宿主同源表达内切葡聚糖酶基因。运用聚合酶链反应(PCR)技术从里氏木霉c DNA中扩增得到内切葡聚糖酶基因cel7b序列,并将其连接到载体p18-m2上构建重组质粒,将重组质粒转化到里氏木霉菌株中,通过筛选获得表达内切葡聚糖酶的重组里氏木霉工程菌。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测显示,发酵液中重组内切葡聚糖酶的分子质量约48 ku。摇瓶发酵结果显示,内切葡聚糖酶在重组里氏木霉中得到分泌表达,发酵液中的内切葡聚糖酶和滤纸酶活分别达到了726 U/mL和28.7 U/mL,分别为出发菌株酶活的2.9倍和1.1倍。玉米芯进行糖化试验结果显示,重组里氏木霉所产酶液糖化玉米芯的酶解得率为81.4%,比出发菌株提高了6.3%。

Using the Trichoderma reesei as host, the endoglucanase gene was homologously expressed. The endoglucanase gene cel7b sequence was amplified by polymerase chain reaction （PCR） from T. reesei cDNA. The amplified products were ligated into the carrier p 18-m2 to construct recom- binant plasmid. The recombinant plasmid was transformed into T. reesei strain, and a recombination T. reesei engineering strain of expressing en- doglucanase was obtained by screening. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the molecular mass of recombination endoglucanase in fermented liquid was about 48 ku. The results of shaking flask fermentation showed that endoglucanase in recombi- nant T. reesei was secreted and expressed. The endoglucanase activity in fermented liquid and filter paper were up to 726 U/ml and 28.7 U/ml, re- spectively, which were 2.9 times and 1.1 times than that of original strain, respectively. The results of saccharification tests of corncob showed that enzymatic hydrolysis yield of corncob saccharified by enzyme from recombinant T. reesei cellulase was 81.4%, which was 6.3% higher than that of original strain.