新型扁桃酸消旋酶的基因挖掘和性能表征

Genome mining and characterization of a new mandelate racemase

摘要:

消旋酶是实现手性化合物去消旋化制备光学纯化学品的重要工具，来源于恶臭假单胞菌的扁桃酸消旋酶(MR)，是目前唯一可以催化两种构型扁桃酸互相转换的消旋酶。通过基因组数据挖掘获得了9个新的扁桃酸消旋酶基因及活性蛋白，其中来源于放射性土壤杆菌Agrobacterium radiobacter的ArMR酶对扁桃酸和邻氯扁桃酸具有较高的催化活力，而且该酶的异源表达水平也较理想。ArMR催化扁桃酸消旋反应的最适温度为50 ℃，最适pH为7.8。该酶在30 ℃、40 ℃和50 ℃下的半衰期分别为70.7、7.2、0.17 h。ArMR对(R)-和(S)-扁桃酸的KM值分别为1.44 mmol/L和0.81 mmol/L，kcat值分别为410 s–1和218 s–1；对(R)-和(S)-邻氯扁桃酸的KM值分别为6.48?mmol/L和6.37 mmol/L，而kcat值为0.22 s–1和0.23 s–1。Mg2+和Mn2+对该酶的活力有促进作用，而Zn2+使其完全失活。新型扁桃酸消旋酶的发现和表征为今后此类酶的深入研究和开发提供了更多资源和数据参考。

Abstract:

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.