α-羟酸多酶同步氧化-还原高效去消旋化体系的构建

Based on the preliminary research results, we select the proprietary stereo-selective α-hydroxyacid dehydrogenase (α-HADH) and ketoacid reductase (KARD) as the objects of this research. We will reveal the key structure area, activity centers and regulation sites of stereoselective α-HADH and KARD on substrates with different substituted groups and locations to illustrate the structure-activity relationship of the enzymes. For different substrates, α-HADH and KARD will be subject to rational design with single-point mutation and multiple-point mutations to generate new mutants exhibiting high stereoselectivity and activity. Many positive mutants towards different substrates will be obtained and the enzymatic characteristics of positive mutants will also be investigated. α-HADH and KARD mutant libraries with high activity and stereoselectivity will be constructed. We will select the appropriate multi-gene co-expression vector construction strategies to optimize α-HADH, KARD and glucose dehydrogenase co-expression system. Through screening efficient enzyme co-expression system and optimizing the reaction conditions, we will establish a production technology platform for chiral α-hydroxy acids. Finally, a new technology for the production of chiral α-hydroxyacids will be developed by the α-hydroxy acid concurrent oxidation-reduction biocatalytic racemization system.

项目以申请者前期筛选的具有知识产权的菌株所产的α-羟酸脱氢酶和酮酸还原酶为研究对象，揭示α-羟酸脱氢酶、酮酸还原酶关键结构区域、活性中心与调控位点，阐明酶的构效关系；针对不同底物，进行α-羟酸脱氢酶、酮酸还原酶的理性设计，对立体选择性、活性具有重要作用的关键位点实施分子改造，构建针对不同取代基团和取代位置的底物立体选择性及活性提高的α-羟酸脱氢酶和酮酸还原酶突变体库，获得针对不同底物立体选择性和活性提高的突变体；对氧化-还原三酶偶联反应的调节与控制及反应机制的有关科学基础问题进行深入的应用基础研究，揭示α-羟酸脱氢酶、酮酸还原酶、葡萄糖脱氢酶表达调控的规律及耦合过程平衡表达水平的规律，建立高效的一菌三酶反应系统，优化同步一菌三酶催化法去消旋化生产手性α-羟酸的高效反应。最终，构建α-羟酸同步氧化-还原生物催化高效去消旋化生产手性α-羟酸的生产技术平台，为手性α-羟酸的生产开辟一条新的途径。

项目以前期筛选的微生物菌株含有的α-羟酸脱氢酶和酮酸还原酶为研究对象，进行了α-羟酸脱氢酶、酮酸还原酶的分子改造；针对不同底物，进行α-羟酸脱氢酶、酮酸还原酶的理性设计，构建了针对不同取代基团和不同取代位置的底物立体选择性和活性提高的α-羟酸脱氢酶和酮酸还原酶突变体库，获得了针对不同底物立体选择性和活性提高的若干个突变体；找到了合适的多基因共表达载体构建策略，进行了α-羟酸脱氢酶、酮酸还原酶、葡萄糖脱氢酶三基因共表达体系的构建优化，对三酶偶联反应的调节与控制及反应机制的有关科学基础问题进行深入研究，建立高效的一菌三酶反应及控制系统。。对于19种外消旋2-羟酸（1a-1m），经过2 h的反应，均被高效地转化为相应的(R)-2-羟酸，收率大于98%，e.e.值大于99%。申请人积累了丰富的多酶催化的经验，利用多酶催化的优势，开发了α-羟酸同步生物催化高效去消旋化生产手性羟酸的技术平台，项目技术还进行了进一步的拓展，发展了氧化-还原去消旋化生产手性氨基酸的技术。