谷氨酸棒杆菌L-天冬氨酸α-脱羧酶在大肠杆菌中的表达及酶转化生产β-丙氨酸  

Expression of L-aspartate α-decarboxylase from Corynebacterium glutamicum in Escherichia coli and its application in enzymatic synthesis of β-alanine

作　　者：赵连真 张梁[1,2] 石贵阳[1,2] 

机构地区：[1]粮食发酵工艺与技术国家工程实验室,江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122

出　　处：《微生物学通报》2013年第12期2161-2170,共10页Microbiology China

基　　金：国家863计划项目(No.2012AA021201;2011AA100905);教育部新世纪优秀人才支持计划项目(No.NCET-11-0665)

摘　　要：【目的】克隆谷氨酸棒杆菌来源L-天冬氨酸α-脱羧酶基因,实现其在大肠杆菌中的异源表达,并进行酶转化L-天冬氨酸合成β-丙氨酸的研究。【方法】PCR扩增谷氨酸棒杆菌L-天冬氨酸α-脱羧酶基因pand,构建表达载体pET24a(+)-Pand,转化宿主菌大肠杆菌BL21(DE3),对重组菌进行诱导表达,表达产物经DEAE离子交换层析和G-75分子筛层析纯化后进行酶学性质研究,然后进行酶转化实验,说明底物和产物对酶转化的影响。【结果】重组菌SDS-PAGE分析表明Pand表达量可达菌体总蛋白的50%以上,AccQ·Tag法检测酶活达到94.16 U/mL。该重组酶最适反应温度为55°C,在低于37°C时保持较好的稳定性,最适pH为6.0,在pH 4.0 7.0范围内有较好的稳定性。酶转化实验说明:底物L-天冬氨酸和产物β-丙氨酸对转化反应均有抑制作用;实验建立了较优的酶转化反应方式,在加酶量为每克天冬氨酸3 000 U时,以分批加入固体底物L-天冬氨酸的形式,使100 g/L底物转化率达到97.8%。【结论】重组L-天冬氨酸α-脱羧酶在大肠杆菌中获得高效表达,研究了酶转化生产β-丙氨酸的影响因素,为其工业应用奠定了基础。[Objective] An L-aspartate α-decarboxylase gene was amplified from Corynebacterium glutamicum and expressed in Escherichia coli, to catalyze L-aspartate to β-alanine. The catalysis characterization of this α-decarboxylase was also studied. [Methods] The Pand gene was amplified from C. glutamicum and cloned into the expression plasmid pET24a（＋）, the recombinant plasmid was transformed into E. coli BL21（DE3）. Pand was then successfully expressed with induction. To explore the enzymatic characteristics of Pand, high purity of Pand was obtained by DEAE ion exchange chromatography and G-75 chromatography. Then the effects of substrate and product on enzymatic conversion were studied. [Results] The Pand constituted more than 50% of the total cell proteins and achieved the activity of 94.16 U/mL analyzed by SDS-PAGE and AccQ·Tag assays respectively. The optimal reaction temperature of the purified Pand was at 55 °C and stable below 37 °C. The optimal reaction pH of Pand was at 6.0 and stable at 4.0？7.0. The enzymatic synthesis was inhibited by L-aspartate and β-alanine. When L-Asp was added in batches with 3 000 U Pand per gram L-Asp, the conversion ratio of 100 g/L L-Asp reached 97.8%. [Conclusion] The L-aspartate α-decarboxylase gene from C. glutamicum was successfully expressed in E. coli.

关 键 词：L-天冬氨酸α-脱羧酶 Β-丙氨酸 谷氨酸棒杆菌 表达 纯化 转化率 

分 类 号：TQ922.2[轻工技术与工程—发酵工程]