4-羟基苯乙酸-3-羟化酶A重组表达菌株的构建及其对羟基酪醇的生物转化研究  

Construction of 4-Hydroxyphenylacetate-3-hydroxylase A Expression Strain and Its Biotransformation Effect on Hydroxytyrosol

作　　者：钱鑫[1] 郭红颜[2] 周庆峰[3] 

机构地区：[1]河南农业大学生命科学学院,郑州450002 [2]上海医药工业研究院,上海200040 [3]商丘师范学院生命科学学院,商丘476000

出　　处：《中国生物工程杂志》2015年第3期56-60,共5页China Biotechnology

基　　金：河南省教育厅重点科技攻关资助项目(14A180008)

摘　　要：目的:构建表达4-羟基苯乙酸-3-羟化酶A(4-hydroxyphenylacetate-3-hydroxylase A,HHA)的重组菌株,进而以酪醇为底物,利用重组菌株转化生产羟基酪醇。方法:选择大肠杆菌BL21(DE3)为模板来扩增HHA基因,经酶切后连接到表达载体p ET-28a中,获得重组表达载体p ET28a-HHA,将重组载体转化到感受态细胞BL21(DE3),在重组菌液中加入适量酪醇,利用胞内的重组酶对酪醇加羟基合成羟基酪醇,细胞离心后,分别采用薄层层析法和气质联用法检测上清液中羟基酪醇的转化结果。结果:IPTG诱导表达后,经SDS-PAGE分析获得分子质量分别为58.8k Da和18.5k Da的两条蛋白质条带。薄层层析法和气质联用法均检测到催化产物羟基酪醇的生成。结论:成功构建了表达HHA的重组菌株,该菌株可有效将酪醇转化为羟基酪醇。In the present study, a recombinant strain that is able to express 4-hydroxyphenylacetate-3-hydroxylase （HHA）. Hydroxytyrosol can be produced in this recombinant strain using tyrosol as the substrate. HHA gene was amplified using E. coli BL21 （DE3） as the template and then connected to the expression vector pET-28a. Obtained recombinant expression vector pET28a-HHA was transformed into competent cell BL21 （DE3）. The suitable amount of tyrosol was used as raw materials to prepare hydroxytyrosol in the recombinant bacteria. Thin layer chromatography and gas chromatography were used respectively to detect the production level of hydroxytyrosol in supernatant. After the IPTG induced, two protein bands （58.8kDa and 18.5kDa） were detected by SDS-PAGE analysis. Hydroxyltyrosol was also detected by thin layer chromatography and gas chromatography. The results showed that the expression recombinant strain HHA was successfully conducted and this strain can effectively convert tyrosol to hydroxytyrosol.

关 键 词：大肠杆菌 酪醇 羟基酪醇 生物转化 

分 类 号：Q81[生物学—生物工程]