苹果酸是各种工业和临床应用的重要材料。枯草芽孢杆菌是一种广泛用于化学生产的生物催化剂工具。然而,枯草芽孢杆菌(BsMDH)苹果酸脱氢酶的特定酶学特性仍然未知。在本研究中,BsMDH被克隆,重组表达和纯化以测试其酶学性质。BsMDH的单个单元的分子量为34,869.7Da。基质辅助激光解吸电离飞行时间质谱和凝胶过滤分析表明,重组BsMDH可以形成二聚体。草酰乙酸和NADH的kcat/Km值高于苹果酸和NAD+,分别,表明在苹果酸合成方向上的催化作用优于相反方向。此外,构建了6个BsMDH突变体,并在辅酶结合位点处替换了氨基酸。其中,BsMDH-T7对NADPH的亲和力和催化效率远高于NADH,变化程度为2039,表明辅酶依赖性从NADH向NADPH转移。此外,BsMDH-T7显示相对较低的Km值,但kcat和kcat/Km高于来自黄热菌和谷氨酸棒杆菌的NADPH依赖性MDHs。总的来说,这些结果表明,BsMDH和BsMDH-T7突变体可能是有前途的苹果酸生产酶。
Malate is an important material to various industrials and clinical applications. Bacillus subtilis is a widely used biocatalyst tool for chemical production. However, the specific enzymatic properties of malate dehydrogenase from Bacillus subtilis (BsMDH) remain largely unknown. In the present study, BsMDH was cloned, recombinantly expressed and purified to test its enzymatic properties. The molecular weight of single unit of BsMDH was 34,869.7 Da. Matrix-Assisted Laser-Desorption Ionization-Time-of-Flight Mass Spectrometry and gel filtration analysis indicated that the recombinant BsMDH could form dimers. The kcat/Km values of oxaloacetate and NADH were higher than those of malate and NAD+, respectively, indicating a better catalysis in the direction of malate synthesis than the reverse. Furthermore, six BsMDH mutants were constructed with the substitution of amino acids at the coenzyme binding site. Among them, BsMDH-T7 showed a greatly higher affinity and catalysis efficiency to NADPH than NADH with the degree of alteration of 2039, suggesting the shift of the coenzyme dependence from NADH to NADPH. In addition, BsMDH-T7 showed a relatively lower Km value, but a higher kcat and kcat/Km than NADPH-dependent MDHs from Thermus flavus and Corynebacterium glutamicum. Overall, these results indicated that BsMDH and BsMDH-T7 mutant might be promising enzymes for malate production.