乳酸乳球菌谷氨酸脱羧酶B重组质粒的构建、异源表达及酶学性质的研究  

Heterologous Expression and Enzymatic Properties,Construction of Lactococcus Lactis Glutamic Acid Decarboxylase B Recombinant Plasmid

作　　者：熊冰钰 林伟朝 梁露露 张毛毛 朱显峰[1] XIONG Bingyu;LIN Weichao;LIANG Lulu;ZHANG Maomao;ZHU Xianfeng(College of Life Sciences,Institute of Biological Engineering,Henan University,Kaifeng 475004,China)

机构地区：[1]河南大学生命科学学院生物工程研究所,河南开封475004

出　　处：《食品科技》2023年第3期1-7,共7页Food Science and Technology

基　　金：河南省科技攻关计划项目(162102210027)。

摘　　要：目的:旨在研究乳酸菌谷氨酸脱羧酶B(Decarboxylation of glutamic acid,gadB)的异源表达及其酶学性质。方法:以Lactocus lactis subsp.lactis Il1403的gadB基因序列为目的基因,实现gadB在大肠杆菌中的异源表达,探究其酶学性质,实现高效催化合成γ-氨基丁酸(γ-Aminobutyric acid,GABA)。结果:重组菌经IPTG诱导gadB过量表达后,进一步采用Ni^(2+)亲和层析纯化gadB,经SDS-PAGE分析,在54 ku处出现明显条带,经检测可溶性蛋白含量约占70%。对酶学性质进行研究,gadB的最适pH值为4.8,最适反应温度为40℃,Na^(+)和Mn^(2+)限制gadB的酶活力,而Mg^(2+)和Zn^(2+)明显刺激酶活。对酶底物浓度进行研究,谷氨酸脱羧酶B的K_(m)为123.83 mmol,V_(max)为0.79 mmol/min。结论:该研究探索了gadB的酶学性质,为基因工程菌工业化生产γ-氨基丁酸提供了实验基础。Objective:The aim was to study the heterologous expression and enzymatic properties of decarboxylation B of glutamic acid(gadB)in lactic acid bacteria.Methods:The gadB gene sequence of Lactocus lactis subsp.Lactis Il1403 was used as the target gene to realize the heterologous expression of gadB in Escherichia coli and explore its enzymatic properties,and to realize efficient catalytic synthesis ofγ-aminobutyric acid(GABA).Results:After overexpression of gadB induced by IPTG,gadB was further purified by Ni^(2+)affinity chromatography.SDS-PAGE analysis showed obvious band at 54 ku,the soluble protein content was about 70%.The optimum pH of gadB was 4.8,and the optimum reaction temperature was 40℃.Na^(+)and Mn^(2+)restricted the enzymatic activity of gadB,while Mg^(2+)and Zn^(2+)stimulated the enzymatic activity obviously.The substrate concentration of glutamate acid decarboxylase B was studied.The K_(m)and V_(max) of glutamate acid decarboxylase B were 123.83 mmol and 0.79 mmol/min,respectively.Conclusion:This study explored the enzymatic properties of gadB and provided an experimental basis for the industrial production ofγ-aminobutyric acid by genetically engineered bacteria.

关 键 词：谷氨酸脱羧酶B Γ-氨基丁酸 重组质粒 异源表达 酶学性质 

分 类 号：TS201.3[轻工技术与工程—食品科学]