β-葡萄糖苷酶与透明颤菌血红蛋白在大肠杆菌中的共表达

Co-expression of β-glucosidase and Vitreoscilla hemoglobin in Escherichia coli

摘要:

为解决大肠杆菌高密度发酵生产β-葡萄糖苷酶过程中溶氧不足的问题，分别采用双顺反子和T7启动子系统在Escherichia coli中引入透明颤菌血红蛋白 (VHb) 以改善溶氧的利用、提高菌体的生物量，进而增加β-葡萄糖苷酶的产量。以双顺反子形式诱导表达VHb时，在摇瓶低溶解氧条件下的最高生物量达到4.24±0.29 (OD600)，与无VHb表达的对照组相比提高了35.03%，同时β-葡萄糖苷酶发酵酶活达到了 (9.78±0.55) U/mL，比对照组提高了25.38%。在3 L发酵罐中使用双顺反子形式共表达VHb时，β-葡萄糖苷酶发酵总酶活达到141.23 U/mL，与无VHb表达的对照相比提高了35.57%。以T7启动子对VHb进行表达后，在摇瓶和发酵罐中β-葡萄糖苷酶的发酵酶活均低于对照组。这些结果表明，在大肠杆菌中以双顺反子形式共表达β-葡萄糖苷酶和VHb能够提升菌体对低溶氧的耐受能力，提高菌体生物量和β-葡萄糖苷酶的产量。

Abstract:

In producing recombinant β-glucosidase in Escherichia coli by high-cell density cultivation (HCDC), insufficient soluble oxygen is always a problem. To address it, Vitreoscilla hemoglobin (VHb) was introduced into Escherichia coli by the bicistron and T7 promoter expression systems, to improve soluble oxygen by bacterial cells and thereby to enhance the biomass and recombinant β-glucosidase production. In the case of bicistron expression system, cell density in shaking flask reached OD600=(4.24±0.29), 35.03% higher than that of the control without VHb. Correspondingly, the maximum activity of β-glucosidase co-expressed with VHb was (9.78±0.55) U/mL, 25.38% higher than that of the control. In a 3-L fermentor, the maximum activity of β-glucosidase was 141.23 U/mL, 35.57% higher than that of the control. In contrast, the activity of β-glucosidase co-expressed with VHb under T7 promoter was lower than that of the control, either in flask or in fermentor. Co-expressing β-glucosidase with VHb using the bicistron expression system may improve the tolerance of E. coli to insufficient soluble oxygen and thus promote the bacterial biomass and the enzyme yield.