Zheng Jian-Yong, Wang Jian, Zhou Sha-Sha, Li Xiao-Jun, Ying Xiang-Xian, Wang Zhao
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, China.
School of Medicine and Life Sciences, Xinyu University, Xinyu, Jiangxi 338004, China.
Protein Expr Purif. 2017 Aug;136:66-72. doi: 10.1016/j.pep.2015.10.001. Epub 2015 Oct 27.
Esterases (EC 3.1.1.X) have been used as biocatalysts due to their good stability, high chemo-, regio- and stereoselectivity. In our previous studies, Bacillus megaterium WZ009 harboring esterase displayed the unique capability to convert (S)-4-Chloro-3-hydroxyethylbutyrate (CHBE) in the racemate to (S)-3-hydroxy-γ-butyrolactone (HL) through stereoselective hydrolysis, dechlorination, and lactonization. The remaining (R)-CHBE and formed (S)-HL could be obtained in a one-pot enzymatic reaction. An esterase from B. megaterium WZ009 was purified and was found to have 466 encoded amino acids and an apparent molecular mass of 55 kDa. The purified esterase exhibited maximal activity at a temperature of 25 °C and at a pH of 11.5 towards 100 mM CHBE. When the stereoselective biocatalysis of rac-CHBE was performed using the recombinant Escherichia coli BL21 (DH3) cells harboring the esterase, the catalytic activity increased by 20-fold compared with the original strain B. megaterium WZ009. With the addition of activated carbon (62 g/L) in the reaction system, the conversion was increased from 39% to 45% at a substrate concentration of 750 mM. Another remarkable advantage is that both of the obtained residual (R)-CHBE and the formed (S)-HL had high optical purities (e.e. > 99.9%, e.e. > 99.9%), thereby making this esterase a usable biocatalyst for industrial application.
酯酶（EC 3.1.1.X）因其良好的稳定性、高化学选择性、区域选择性和立体选择性而被用作生物催化剂。在我们之前的研究中，携带酯酶的巨大芽孢杆菌WZ009表现出独特的能力，可通过立体选择性水解、脱氯和内酯化反应，将外消旋体中的（S）-4-氯-3-羟基丁酸乙酯（CHBE）转化为（S）-3-羟基-γ-丁内酯（HL）。剩余的（R）-CHBE和生成的（S）-HL可在一锅酶促反应中获得。从巨大芽孢杆菌WZ009中纯化得到一种酯酶，发现其编码466个氨基酸，表观分子量为55 kDa。纯化后的酯酶在25℃、pH为11.5时对100 mM CHBE表现出最大活性。当使用携带该酯酶的重组大肠杆菌BL21（DH3）细胞进行rac-CHBE的立体选择性生物催化时，催化活性比原始菌株巨大芽孢杆菌WZ009提高了20倍。在反应体系中加入62 g/L的活性炭后，底物浓度为750 mM时，转化率从39%提高到了45%。另一个显著优点是，所获得的剩余（R）-CHBE和生成的（S）-HL均具有高光学纯度（对映体过量值>99.9%，对映体过量值>99.9%），因此该酯酶成为一种可用于工业应用的生物催化剂。
