嗜热菌β-葡萄糖苷酶A基因的克隆表达及转化大豆异黄酮糖苷  

Cloning and expression β-glucosidase A gene from Thermoanaerobacter ethanolicus for conversion of soybean isoflavone glycosides

作　　者：王锐丽[1] 孙伟[1] 薛业敏[2] 

机构地区：[1]信阳农林学院生物与制药工程学院,河南信阳464000 [2]南京师范大学金陵女子学院,江苏南京210097

出　　处：《中国酿造》2016年第3期114-119,共6页China Brewing

基　　金：河南省科技攻关资助项目(132102110047)

摘　　要：对嗜热厌氧乙醇菌（Thermoanaerobacter ethanolicus）JW200基因组DNA中β-葡萄糖苷酶A基因（Te-BglA）进行PCR扩增，构建重组质粒pET-20b-Te-BglA，并对纯化的重组酶Te-BglA的酶学性质和动力学参数进行研究。结果表明，基因Te-BglA在Escherichiacoli细胞中成功表达，获得重组酶Te-BglA，该酶的最适温度和pH值分别为80℃和7．0。以对硝基苯酚-β-葡萄糖苷（pNPG）为底物时，Km值为（0．78±0．02）mmol／L，Kcat/Km值为（6．86±0．16）×10^4／（mol·s）；以天然底物水杨苷为底物时，Km值为（5．18±0．10）mmol／L，Kcat／Km值为（2．04±0．02）×10^4L／（mol·s），两者比较可知，重组酶Te-BglA表现出对pNPG更好的亲和力和更高的催化效率常数。该酶在80℃保温1h后相对于冰浴中（未保温）的酶活仍有70．1％的残留，在pH4．5～8．0区间仍保持较高的pH稳定性。酶解大豆粉的结果表明，重组酶Te-BglA在80℃作用3h后，大豆黄素和染料木素的产量分别达到35．9mg／g和59．1mg／g。In order to obtain a high stability and efficiency β-glucosidase A （BglA） in the biotransformation of isoflavone glycosides,β-glucosidase gene was amplified by PCR from the genomic DNA of Thermoanaerobacter ethanolicus JW200, and produced in Escherichia coli JM109（DE3） and purified by nickel affinity chromatography for characterization of their enzymatic and kinetic properties, p-nitrophenyl-β-D-glucoside（pNPG） as sub- strate, optimal temperature and pH of the BglA were 80 ℃ and 7.0, Km and Kcat/Km of BglA were （0.78±0.02） mmol/L and （6.86±0.16）× 10^4 L/（mol · s）, respectively. The salicin as substrate, Km and Kcat/Km were （5.18±0.10） mmol/L and （2.04±0.02）×10^4 L/（mol · s）, which demonstrated that the recombi- nant Te-BglA showed better affinity for pNPG and higher catalytic efficiency constant. And BglA was found to be greater stability which kept 70.1% activity of 1 h at 80 ℃ and pH 4.5-8.0. High performance liquid chromatography （HPLC） results demonstrated that the effect of enzyme hydrolysis soybean isofavone glycosides to isoflavone aglycones was evident at 80 ℃ for 3 h, the yield of soybean daidzein and genistein reached 35.9 mg/g and 59.1 mg/g.

关 键 词：Β-葡萄糖苷酶 酶学性质 大豆异黄酮糖苷 苷元 

分 类 号：Q93[生物学—微生物学]