α-半乳糖苷酶基因在大肠杆菌中的表达及酶学性质研究  

Expression and enzymatic properties of α-galactosidase gene in Escherichia coli

作　　者：刁文涛[1,2] 向凌云[1,2] 宁萌[1,2] 王雪妍[1,2] 马焕[1] 冯菲[1,2] 陈国参[1,2] 周伏忠[1,2] DIAO Wentao;XIANG Lingyun;NING Meng;WANG Xueyan;MA Huan;FENG Fei;CHEN Guocan;ZHOU Fuzhong(Institute of Biology Co.,Ltd.,Henan Academy of Sciences,Zhengzhou 450008,China;Key Laboratory of Microbial Engineering of Henan Province,Zhengzhou 450008,China)

机构地区：[1]河南省科学院生物研究所有限责任公司,河南郑州450008 [2]河南省微生物工程重点实验室,河南郑州450008

出　　处：《中国酿造》2019年第8期60-66,共7页China Brewing

基　　金：河南省科学院2018重大科技突破专项（18ZP05001）;河南省科学院基本科研业务费（18JK16011）;河南省科学院助推科技成果转化专项（18ZT05017）

摘　　要：利用大肠杆菌(Escherichia coli)表达系统,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导,成功异源表达了一株链球菌(Streptococcus)S1的α-半乳糖苷酶基因,重组α-半乳糖苷酶经镍柱纯化后,测定其酶学性质。重组α-半乳糖苷酶最适pH值为6.5,最适温度为50℃,在碱性环境中(pH 7.5~10.0)及在40℃以下温度条件下该酶较为稳定;酶催化动力学结果显示,该酶在最适条件下水解硝基苯-α-D-半乳糖苷(pNPG)的最大水解速率(Vmax)为508.38μmol/(min·mg),米氏常数(Km)值为1.2 mmol/L;通过薄层层析(TLC)法检测到该重组α-半乳糖苷酶可以高效地水解天然底物蜜二糖、棉籽糖和水苏糖中的α-半乳糖苷键。The recombinantα-galactosidase gene from Streptococcus S1 was successfully heterologous expressed in Escherichia coli induced by isopropyl-β-D-thiogalactoside(IPTG).The recombinantα-galactosidase was purified by nickel column and its enzymatic properties were determined.The optimal pH value and temperature of the recombinantα-galactosidase were 6.5 and 50℃,respectively,and the enzyme was stable at pH 7.5-10.0 and below 40℃.The results of enzymatic kinetic showed that the maximum hydrolysis rate(Vmax)of p-nitrophenyl-α-D-galactoside(pNPG)hydrolyzed by the enzyme was 508.38μmol/(min·mg),and the Michaelis constant(Km)value was 1.2 mmol/L under the optimal conditions.The results of thin layer chromatography(TLC)showed that recombinantα-galactosidase could efficiently hydrolyzeα-galactoside bond in the natural substrates melibiose,raffinose and stachyose.

关 键 词：链球菌 大肠杆菌 异源表达 重组α-半乳糖苷酶 酶学性质 

分 类 号：Q556.2[生物学—生物化学]