分散泛菌蔗糖异构酶在大肠杆菌中的表达及发酵优化

为了提高分散泛菌Pantoea dispersa UQ68J来源的蔗糖异构酶产量,研究了不同信号肽及发酵条件对蔗糖异构酶在大肠杆菌中重组表达的影响。将携带天然信号肽的蔗糖异构酶基因优化后,转入大肠杆菌Escherichia coli BL21(DE3)构建重组表达菌株——ORI菌株,摇瓶发酵总酶活和胞外酶活分别为85 U/m L、65 U/m L。从天然信号肽开始第22位氨基酸作为成熟蛋白的起始,连接Pel B或Omp A信号肽构建P22和O22菌株,其中P22菌株发酵总酶活提高至138 U/m L,是ORI菌株总酶活的1.6倍;而O22菌株发酵总酶活和ORI菌株无明显差别。采用3.0 g/L的乳糖诱导,P22菌株的蔗糖异构酶总酶活提高至168 U/m L。在3 L发酵罐中,研究甘氨酸浓度和诱导时间对蔗糖异构酶分泌的影响,当补加0.5%甘氨酸,DCW为18 g/L(OD_(600)=30)开始诱导,P22菌株的蔗糖异构酶胞外酶活最高达1 981 U/m L,同时蔗糖异构酶总酶活达到2 640 U/m L,是已报道大肠杆菌重组表达蔗糖异构酶的最高水平。

Expression and production optimization of sucrose isomerase from Pantoea dispersa in Escherichia coli

To improve the yield of sucrose isomerase from Pantoea dispersa UQ68J, we studied the effect of different signal peptides and fermentation conditions on sucrose isomerase expression in Escherichia coli. The gene of sucrose isomerase was optimized and expressed in E. coli BL21 (DE3) with native signal peptide which was named as ORI strain. The total and extracellular enzyme activity was 85 and 65 U/mL in the flask, respectively. The mature protein, which started from the 22th amino acid, was connected with the PelB and OmpA signal peptide to construct P22 and O22 strain, respectively. The total activity of P22 reached 138 U/mL, which was 1.6 times of ORI strain. The total activity of O22 strain was similar to that of ORI strain. Induced by 3.0 g/L lactose, the total activity of P22 strain increased to 168 U/mL. In 3 L fermentor, the effects of glycine concentration and induction time were studied. Induction when the DCW reached 18 g/L (OD600=30), with 0.5% glycine, the extracellular enzyme activity reached 1 981 U/mL, and the total enzyme activity reached 2 640 U/mL, which is the highest activity of sucrose isomerase that was expressed in recombinant E. coli.