新型NAD非依赖性D-乳酸脱氢酶催化机理及应用基础研究

NAD非依赖性乳酸脱氢酶（iLDH）涉及微生物致病过程、2-酮基羧酸与手性羟基羧酸的生物催化法生产。申请者前期在假单胞菌乳酸代谢及L-iLDH的应用基础研究过程中发现，施氏假单胞菌SDM中存在一种新型D-iLDH。与已报道的D-iLDH相比，SDM中新型D-iLDH除含一FAD结合结构域外，另含一铁硫簇结合结构域，因而其催化机理与电子传递过程可能具有独特之处。本申请拟以菌株SDM为研究对象，敲除D-iLDH编码基因，确定新型D-iLDH在SDM菌株D-乳酸代谢中的关键作用；外源表达并分离纯化该新型D-iLDH，通过圆二色谱、电子顺磁共振、等温滴定量热及生物大分子相互作用分析等技术确定D-iLDH各结构域的生物学功能；分析该新型D-iLDH获取及传递电子的具体过程，阐明该新型D-iLDH催化D-乳酸氧化的具体机理；为拓展D-iLDH在2-酮基羧酸与手性羟基羧酸生产方面的应用奠定理论基础。

NAD-independent lactate dehydrogenase (iLDH) involves in the microbial pathogenic process, biocatalytic production of 2-keto carboxylic acids and chiral 2-hydroxy carboxylic acids. In our previous research related to Pseudomonas lactate metabolism and L-iLDH application, a new D-iLDH was found in Pseudomonas stutzeri SDM. Compared with the reported D-iLDH which only contains an FAD-binding domain, the D-iLDH in P. stutzeri SDM also contains an iron-sulfur cluster-binding domain. Thus, the catalytic mechanism and electron transfer process of the novel D-iLDH may be different from the reported D-iLDHs. In this work, the encoding gene of the D-iLDH would be knockout and the critical role of the new D-iLDH in D-lactate metabolism of P. stutzeri SDM would be identified. After exogenous expression and purification of the new D-iLDH, the biological function of each domain of the D-iLDH would be identified by circular dichroism chromatography, electron paramagnetic resonance, isothermal titration calorimetry and surface plasmon resonance. Based on analysis of the electron acquisition and transfer process, the catalytic mechanism of the new D -iLDH would be clarified. This work would also lay the theoretical foundation to expand the application of D-iLDH in the 2-keto carboxylic and chiral 2-hydroxy carboxylic acids production.