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

Sortase A-mediated oligomers of (S)-carbonyl reductase Ⅱ suitable for biotransformation of (S)-phenyl-1, 2-ethanediol

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1 h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3 h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sortase A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。

Abstract:

[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)-1-phenyl-1,2-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-1,2-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)-1-phenyl-1,2-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.