Zhou Maozhi, Tang Cunduo, Xu Jianhe, Yu Huilei
State Key Laboratory of Bioreactor Engineering, School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
Henan Provincial Engineering Laboratory of Insect Bio-reactor, Nanyang Normal University, Nanyang 473061, Henan, China.
Sheng Wu Gong Cheng Xue Bao. 2018 Jun 25;34(6):897-905. doi: 10.13345/j.cjb.170441.
Racemases have been applied for the synthesis of enantiomerically pure compounds through the deracemization methods. Mandelate racemase from Pseudomonas putida was the only enzyme that catalyzes the interconversion of mandelate enantiomers. Using genome mining approaches, we identified 9 mandelate racemases (MRs). A novel racemase named ArMR with higher activity and better soluble protein expression, was isolated from Agrobacterium radiobacter. ArMR displayed the optimum catalytic activity at 50 ℃, pH 7.8 in Tris-HCl buffer. The half-life of ArMR at 50, 40 and 30 ℃ was 0.17, 27.2 and 70.7 h, respectively. KM parameter of ArMR towards (R)- and (S)-mandelic acid was 1.44 and 0.81 mmol/L, respectively; the corresponding kcat value was 410 s⁻¹ and 218 s⁻¹. In addition, KM of ArMR towards (R)- and (S)-2-chloro mandelic acid was 6.48 and 6.37 mmol/L, and the corresponding kcat value 0.22 s⁻¹ and 0.23 s⁻¹, respectively. Meanwhile, Mg²⁺ and Mn²⁺ could activate the enzyme whereas Zn²⁺ inactivated the enzyme completely. Discovery of more novel MRs provides supports further research and development of these racemases.
消旋酶已通过外消旋化方法用于合成对映体纯的化合物。恶臭假单胞菌的扁桃酸消旋酶是唯一催化扁桃酸对映体相互转化的酶。利用基因组挖掘方法，我们鉴定出了9种扁桃酸消旋酶（MRs）。从放射土壤杆菌中分离出一种具有更高活性和更好可溶性蛋白表达的新型消旋酶ArMR。ArMR在Tris-HCl缓冲液中，50℃、pH 7.8时表现出最佳催化活性。ArMR在50℃、40℃和30℃下的半衰期分别为0.17小时、27.2小时和70.7小时。ArMR对（R）-和（S）-扁桃酸的KM参数分别为1.44和0.81 mmol/L；相应的kcat值分别为410 s⁻¹和218 s⁻¹。此外，ArMR对（R）-和（S）-2-氯扁桃酸的KM分别为6.48和6.37 mmol/L，相应的kcat值分别为0.22 s⁻¹和0.23 s⁻¹。同时，Mg²⁺和Mn²⁺可激活该酶，而Zn²⁺则完全使该酶失活。更多新型MRs的发现为这些消旋酶的进一步研究和开发提供了支持。
