【摘要】 厌氧真菌能够产生高比活力的纤维素酶,但由于对厌氧环境的生产条件要求严格,生长速度缓慢,并不适合规模生产。本研究将厌氧真菌内切型-β-葡聚糖酶基因af1与大肠杆菌表达载体pET-28a(+)连接,得到重组质粒pET-28a(+)-af1。以大肠杆菌BL21(DE3)为宿主,重组质粒转化后获得重组大肠杆菌BL21(DE3)/pET-28a(+)-af1。采用SDS-PAGE蛋白电泳对重组大肠杆菌的表达产物进行分析,在40 kDa附近得到与目的基因af1表达蛋白理论值相当的明显条带。AF1酶蛋白的最适pH为6.0,最适温度为45°C。重组大肠杆菌的摇瓶产酶试验结果显示:诱导培养18 h时,内切型-β-葡聚糖酶(CMC酶)活力可达410 U/mL。该项研究工作为进一步构建内切型-β-葡聚糖酶高产菌株奠定了良好的基础。更多还原

【Abstract】 Anaerobic fungus can produce high specific activity cellulase. However, its large- scale production is limited due to strict anaerobic processing conditions and low growth rate. In this study, an endo- β- glucanase(CMCase) gene af1 from anaerobic fungus was inserted into Escherichia coli expression vector pET-28a(+). Then the recombinant plasmid pET-28a(+)-af1 was transformed into Escherichia coli BL21(DE3) to obtain recombinant strain BL21(DE3)/pET- 28a(+)- af1. SDS- PAGE analysis of its expression product showed a clear protein band near 40 kDa, which accorded with that of AF1 protein theoretic size. The AF1 enzyme exhibited maximum CMCase activity at 45 °C, pH 6.0.Shaking flask experiment demonstrated that, the CMCase activity of AF1 could reach 410 U/mL at 18 h after induction. The research result is useful for further work involving obtaining a high endo- β-glucanase producing strain.更多还原