β-半乳糖苷酶和阿拉伯糖异构酶共表达一步法催化乳糖到塔格糖

One-step synthesis of lactose to tagatose by co-expressing β-galactosidase and arabinose isomerase

摘要:

[目的] 构建一株以廉价原料乳糖为底物合成塔格糖的重组菌株，实现一步法高效生物合成稀有糖——塔格糖。[方法] 从Escherichia coli K-12基因组中，PCR扩增出阿拉伯糖异构酶araA和β-半乳糖苷酶lacZ基因，以SD-AS为连接子，利用pET28a-1载体串联表达于Escherichia coli BL21（DE3），获得重组菌E.coli BL21/pET28a-araA-lacZ，对重组菌全细胞催化合成塔格糖的条件进行了工艺优化与放大研究。[结果] araA和lacZ基因在E.coli BL21中同时高效表达，在最优条件（pH 8.0、温度50℃、5 mmol/L Mn²⁺、添加0.5 mol/L硼酸和0.1% SDS）下，E.coli BL21/pET28a-araA-lacZ全细胞转化100 g/L乳糖，合成塔格糖最高产量达24.03±2.03 g/L，乳糖到塔格糖的摩尔转化率为45.67%，随着底物乳糖浓度的提高，塔格糖产量呈不同程度的提高，当投加500 g/L底物乳糖时，全细胞合成塔格糖产量最高达83.81±1.38 g/L。[结论] 通过2个关键靶酶的编码基因araA和lacZ在E.coli BL21细胞中进行共表达，实现了以重组菌全细胞为催化剂转化廉价底物乳糖，一步法高效合成稀有糖塔格糖，该研究为生物法制备低能量的功能性稀有糖奠定了较好的研究基础。

Abstract:

[Objective] To achieve the high-efficiency synthesis of lactose to tagatose in one-step, we cloned β-galactosidase gene (lacZ) and arabinose isomerase gene (araA) from Escherichia coli K-12 genome and co-expressed in E. coli BL21(DE3).[Methods] The araA and lacZ genes were amplified from the E. coli K-12 genome by PCR. The two genes with SD-AS sequence as a linker were cloned into the expression vector pET28a-1 to get the recombinant plasmid pET28a-araA-lacZ, which was transformed into the competent cells of E. coli BL21(DE3) to obtain E. coli BL21/pET28a-araA-lacZ. The synthesis conditions of tagatose by whole cells of E. coli BL21/pET28a-araA-lacZ were optimized and the process was scaled up.[Results] The araA and lacZ genes were efficiently co-expressed in E. coli BL21 simultaneously. The optimal conditions for the synthesis of tagatose by the whole cells of E. coli BL21/pET28a-araA-lacZ were determined:pH 8.0, 50℃, 5 mmol/L Mn²⁺, 0.5 mol/L borate and 0.1% SDS as permeabilizing agent. Under these optimal conditions, the highest yield of tagatose was 24.03±2.03 g/L with a molar conversion rate of 45.67% using 100 g/L lactose as substrate. The yield of tagatose was increased with the increasment of the substrate lactose concentration. The yield of tagatose was 83.81±1.38 g/L with 500 g/L lactose as substrate.[Conclusion] The genes lacZ and araA coding two target enzymes were co-expressed in E. coli BL21 to realize the efficient synthesis of high valued rare sugar tagatose from the cheap substrate lactose in one step. This research has laid a good research foundation for the preparation of low-energy functional sugars by biological methods.