Sortase A介导的(S)-羰基还原酶Ⅱ寡聚体高效立体选择性转化(S)-苯基乙二醇

摘要

[目的]以金黄色葡萄球菌(Staphylococcus aureus)的sortase A为“分子订书机”,用于(S)-羰基还原酶Ⅱ分子之间的连接,获得催化功能与稳定性增强的氧化还原酶寡聚体,高效催化2-羟基苯乙酮,合成(S)-苯基乙二醇.[方法]从S.aureus基因组中克隆sortase A基因,在大肠杆菌中表达,通过镍柱和凝胶层析纯化重组酶,获得纯酶sortase A.通过基因工程手段在(S)-羰基还原酶Ⅱ的C末端添加GGGGSLPETGG序列,蛋白纯化获得(S)-羰基还原酶Ⅱ-GGGGSLPETGG,摸索了sortase A催化(S)-羰基还原酶Ⅱ-GGGGSLPETGG的分子连接,形成(S)-羰基还原酶Ⅱ寡聚体的最佳条件,并研究了寡聚体酶学性质及生物转化(S)-苯基乙二醇的效率.[结果](S)-羰基还原酶Ⅱ寡聚体比酶活力为38.5 U/mg,比原始型(S)-羰基还原酶Ⅱ提高了6倍,最适反应温度为50℃,最适pH为6.0,在50℃放置1h后酶活仍旧保持90％以上;蛋白质变性实验结果显示,(S)-羰基还原酶Ⅱ寡聚体的变性温度为60.1℃,比原始酶提高了10℃;生物转化结果显示(S)-羰基还原酶Ⅱ寡聚体在3h内完全转化5 g/L 2-羟基苯乙酮,产生光学纯度为100％的(S)-苯基乙二醇,相比于重组大肠杆菌(S)-羰基还原酶Ⅱ全细胞催化时间缩短了16倍.[结论]本研究首次将sortaseA应用于氧化还原酶的分子连接,显著提高了酶的催化效率和热稳定性,表明sortase在手性催化中有很大的潜在应用价值.%[Objective] To obtain oligomers of (S)-carbonyl reductase Ⅱ with strong activity and stability,we used sortase A from Staphylococcus aureus as molecular "stapler" to conjugate (S)-carbonyl reductase Ⅱ.The (S)-carbonyl reductase Ⅱ oligomers efficiently catalyzed the biotransformation of 2-hydroxyacetophenone to (S)-l-phenyl-l,2-ethanediol.[Methods] We cloned sortase A gene from S.aureus genome and expressed it in Escherichia coli.The recombinant enzyme was purified through Ni-affinity and gel filtration chromatography.Meanwhile,we added GGGGSLPETGG to C terminus of (S)-carbonyl reductase Ⅱ using genetic techniques,and purified recombinant SCRⅡ-GGGGSLPETGG to homogeneity.We determined the optimal reaction conditions of sortase A-mediated ligation of (S)-carbonyl reductase Ⅱ to oligomers.The enzyme characteristics of the generated oligomers were studied.And the oligomers-catalyzed biotransformation efficiency of (S)-1-phenyl-l,2-ethanediol was further detected.[Results] Oligomers showed a specific activity of 38.5 U/mg,6-fold increase compared to (S)-carbonyl reductase Ⅱ.The optimal temperature and pH of ligation reaction by oligomers were 35 ℃ and 6.0 respectively.The relative activity was maintained over 90％ at 50 ℃ for 1 hour.Denaturation test showed that the denaturation temperature of oligomers was 60.1 ℃,10 ℃ higher than that of (S)-carbonyl reductase Ⅱ.Biotransformation results indicated that oligomers completely transformed 5 g/L 2-hydroxyacetophenone within 3 hours to generate (S)-l-phenyl-l,2-ethanediol with an optical purity of 100％.With oligomers,we reduced transformation duration for 16 folds compared to that of recombinant E.coli-(S)-carbonyl reductase Ⅱ cells.[Conclusion] This work first described sortase A-mediated the ligation of oxidoreductase and significantly improved catalytic efficiency and thermal stability of enzyme,suggesting sortase had great potential application in chiral synthesis.