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-expressingβ-galactosidase and arabinose isomerase

作　　者：李志月 张显[1] 饶志明[1,2] 张荣珍 Zhiyue Li;Xian Zhang;Zhiming Rao;Rongzhen Zhang(Key Laboratory of Industrial Biotechnology,Ministry of Education,School of Bioengineering,Jiangnan University,Wuxi 214122,Jiangsu Province,China;National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University,Wuxi 214122,Jiangsu Province,China)

机构地区：[1]江南大学生物工程学院,教育部工业生物技术重点实验室,江苏无锡214122 [2]江南大学粮食发酵工艺与技术国家工程实验室,江苏无锡214122

出　　处：《微生物学报》2021年第9期2907-2920,共14页Acta Microbiologica Sinica

基　　金：国家重点研究发展计划(2018YFA0900300);国家自然科学基金(31970045);国家轻工业技术与工程一流学科计划(LITE2018-12);高等学校学科创新引智计划(111-2-06);江苏省高校学术计划;宁夏回族自治区重点研发计划(2020BFH02011)。

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 Mn^(2+)、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。[Objective]To achieve the high-efficiency synthesis of lactose to tagatose in one-step,we clonedβ-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 Mn^(2+),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.

关 键 词：Β-半乳糖苷酶 阿拉伯糖异构酶 塔格糖 乳糖 生物催化 

分 类 号：Q78[生物学—分子生物学]