新颖深海微生物酯酶EstC11的酶学性质及其在手性拆分乙酸苏合香酯中的应用

Characterization of a novel deep-sea microbial esterase EstC11 for enantioselective resolution of(±)-1-phenylethl acetate

【背景】手性乙酸苏合香酯是重要的手性香料产品,在食品及精细化工等领域都有重要的应用。酶催化不对称合成手性乙酸苏合香酯产品具有极好的工业应用前景。【目的】研究酯酶EstC11的基本酶学性质及其在制备手性乙酸苏合香酯中的应用。【方法】对来自西太平洋深海热液口芽孢杆菌Bacillus sp.CX01中的新颖微生物酯酶基因EstC11进行克隆、表达及酶学性质鉴定。通过对p H、温度、有机溶剂等反应条件的优化提高酯酶手性拆分乙酸苏合香酯的光学纯度。【结果】酯酶EstC11的最适反应p H为8.5,最适温度为25°C,一些金属离子和有机溶剂对酯酶EstC11的水解活性具有不同程度的抑制作用。通过对反应条件的优化,在最适反应条件下(p H 9.0 50 mmol/L Tris-HCl,20°C,50 mmol/L底物浓度)反应3 h后,(R)-乙酸苏合香酯的光学纯度达98%,得率为39%。【结论】通过对酯酶拆分条件的优化,手性拆分乙酸苏合香酯生成(R)-乙酸苏合香酯的光学纯度明显提高,为酯酶EstC11在工业化上的应用奠定了基础。

[Background] Chiral phenylethl acetate is a crucial chiral flavor compound and possesses important applications in the fields of food and fine chemicals. The enzymatic synthesis of chiral phenylethl acetate is of great industrial application prospect. [Objective] To characterize a novel microbial esterase Est C11 for enantioselective resolution of（±）-1-phenylethl acetate. [Methods] A microbial esterase Est C11 identified from the deep sea of Western Pacific Ocean was cloned, expressed, and characterized. The optical purity of chiral product（R）-1-phenylethl acetate generated through enzymatic kinetic resolution was increased by optimizing the conditions of enzymatic reactions, such as p H, temperature and organic solvents. [Results] The optimum p H and temperature of Est C11 were 8.5 and 25 °C, respectively. Some metal ions and organic solvents could inhibit the hydrolytic activity of Est C11. Under the optimum reaction conditions（p H 9.0, 50 mmol/L Tris-HCl, 20 °C, 50 mmol/L substrate concentration）, the optical purity of（R）-1-phenylethl acetate reached 98% with the yield of 39% after 3 h. [Conclusion] The optical purity and yield of（R）-1-phenylethl acetate were dramatically improved after the optimization of enzymatic reaction conditions. Thus, our study laid the foundation for the application of esterase Est C11 in industry.