埃及伊蚊来源半胱亚磺酸脱羧酶的性质解析  

Characterization of cysteine sulfinic decarboxylase from Aedes aegypti

作　　者：郑文慧 刘佳伟 刘子铭 周哲敏[1] 刘中美[1] ZHENG Wenhui;LIU Jiawei;LIU Ziming;ZHOU Zhemin;LIU Zhongmei(School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与发酵工业》2021年第18期30-36,共7页Food and Fermentation Industries

基　　金：国家重点研发计划项目(2017YFE0129600);国家自然科学基金面上项目(21878125)。

摘　　要：原核来源L-天冬氨酸-α-脱羧酶的活性较低,是限制生物法合成β-丙氨酸的主要因素,该研究将目光由原核来源转向真核来源,意在寻找具有更高催化活性的酶。该文通过基因合成获得埃及伊蚊来源半胱亚磺酸脱羧酶,并添加sumo标签实现了其在大肠杆菌中的高效表达,酶学性质表征实验结果表明,重组酶可催化L-天冬氨酸生成β-丙氨酸,比酶活力为(5.00±0.074)U/mg,添加sumo标签后比酶活力为(5.40±0.129)U/mg;最适反应条件为40℃,pH 6.0;带标签酶的稳定性要明显优于野生型,在50℃处理30 min后酶活力仍保留40%,在pH 5.0~8.0酶活力都维持在80%左右。利用大肠杆菌自身的天冬氨酸裂解酶与重组半胱亚磺酸脱羧酶相偶联,实现了从富马酸到天冬氨酸的一步转化,产物的摩尔转化率达到93.03%。该研究中半胱亚磺酸脱羧酶对于L-天冬氨酸具有较高的催化活性,为β-丙氨酸的工业化生产提供理论支持和技术参考。Prokaryotic source L-aspartate-α-decarboxylase has low activity,which is the main factor restricting the biosynthesis ofβ-alanine.In this study,we shifted our attention from prokaryotic sources to eukaryotic sources,and aimed to find higher catalytically active enzymes.We obtained cysteine sulfinic decarboxylase from Aedes aegypti through gene synthesis,and added the sumo tag to achieve high-efficiency expression in Escherichia coli.The results of enzymatic characterization experiments showed that the recombinant enzyme could catalyze L-aspartic acid to produceβ-alanine,the specific enzyme activity was(5.60±0.074)U/mg,after adding the sumo tag,the specific enzyme activity was(5.80±0.129)U/mg.The optimal reaction conditions were 40℃and pH 6.0.The stability of the tagged enzyme was significantly better than that of the wild type.The enzyme activity still retained 40%after treatment at 50℃for 30 min,and the enzyme activity remained at about 80%between pH 5.0-8.0.The cysteine sulfinic decarboxylase was coupled with L-aspartic acid lyase of E.coli to realize the one-step conversion from fumaric acid to aspartic acid,and the molar conversion rate of the product reached 93.03%.In this study,cysteine sulfinic decarboxylase had high catalytic activity for L-aspartic acid,which provided technical reference and theoretical support for the industrial production ofβ-alanine.

关 键 词：半胱亚磺酸脱羧酶 L-天冬氨酸 Β-丙氨酸 酶学性质 全细胞催化 

分 类 号：Q814.9[生物学—生物工程]