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

作　　者：周茂志 唐存多[2] 许建和[1] 郁惠蕾[1] Maozhi Zhou;Cunduo Tang;Jianhe Xu;and Huilei Yu(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-reaetor, Nanyang Normal University, Nanyang 473061, Henan, China)

机构地区：[1]华东理工大学生物工程学院生物反应器工程国家重点实验室,上海200237 [2]南阳师范学院昆虫生物反应器河南省工程实验室,河南南阳473061

出　　处：《生物工程学报》2018年第6期897-905,共9页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.21536004)资助~~

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Ar MR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K_M值分别为1.44 mmol/L和0.81 mmol/L,k_(cat)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k_(cat)值为0.22 s^(–1)和0.23 s^(–1)。Mg^(2+)和Mn^(2+)对该酶的活力有促进作用,而Zn^(2+)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。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 Ar MR with higher activity and better soluble protein expression, was isolated from Agrobacterium radiobacter. Ar MR displayed the optimum catalytic activity at 50 ℃, pH 7.8 in Tris-HCl buffer. The half-life of Ar MR at 50, 40 and 30 ℃ was 0.17, 27.2 and 70.7 h, respectively. K_M parameter of Ar MR towards（R）-and（S）-mandelic acid was 1.44 and 0.81 mmol/L, respectively; the corresponding k_（cat）value was 410 s^（–1） and 218 s^（–1）. In addition, KM of Ar MR towards（R）-and（S）-2-chloro mandelic acid was 6.48 and 6.37 mmol/L, and the corresponding k_（cat） value 0.22 s^（–1） and 0.23 s^（–1）, respectively. Meanwhile, Mg^（2＋） and Mn^（2＋） could activate the enzyme whereas Zn^（2＋） inactivated the enzyme completely. Discovery of more novel MRs provides supports further research and development of these racemases.

关 键 词：扁桃酸消旋酶 扁桃酸 邻氯扁桃酸 基因挖掘 催化性能 动力学常数 

分 类 号：Q78[生物学—分子生物学] TQ460.1[化学工程—制药化工]