分子定向固定化NADH氧化酶的研究

The regeneration of cofactors is a very crucial step for enzymatic redox reactions because the use of these cofactors in stoichiometric amounts is too expensive. NADH oxidase is considered as one of the best enzyme for regeneration of NAD because there is no by-products, no additives, and high biocompaticity in the regeneration reaction. In our previous work, a water-forming NADH oxidase from Lactobacillus rhamnosus (LrNox) has been cloned and expressed in E. coli BL21 sucessfully. The bottleneck for appliaction of NADH oxidase is its immobilization. In this proposal, we try to immobilize LrNox on nanoparticles for in situ regeneration of NAD. Firstly, we will modify LrNox based on its structure by mutagenesis for orientated immobilization. The modification area should be the surface region far away from catalytic center to maintain the catalytic activity of the enzyme. The modified site should also be included inside the area which Lys residuals are rich for multiple-point covalent immobilization. The modification will introduce Cys residual for orientation onto the carrier. Secondly, the nanomagnetic materials, Fe3O4@SiO2 core-shell nanoparticles with various functional groups including aldehyde, oxirane and disulfate will be prepared to react with Cys and Lys on the enzyme surface. Finally, the immobilization will be performed with a two-step reactions: enzyme will be site-directed immobilized onto the magnetic nanoparticles by thiol-disulfate exchanges; then the Lys will react with functional groups (oxirane or aldehyde etc.) on the carrier for multiple-points covalent attachment. The immobilized LrNox will be employed for in situ NAD regeneration in production of chiral drugs. It has advantages such as low diffusional limitation and easy to recovery. This reasearch will help the design of enzyme immobilization with protein techniques to control the orientation, and also favor the development of cofactor regeneration.

辅酶再生是酶催化氧化还原反应工业化的瓶颈，NADH氧化酶被认为是辅酶NAD再生最有应用前景的酶，关键是如何将其高效地固定化，而分子定向固定化是迄今极具吸引力的固定化方法。本项目以高效定向固定化NADH氧化酶为目标，从分子定点突变改造、磁性纳米载体制备、酶与载体定向固定三个方面开展研究工作。首先基于前期研究中克隆到的NADH氧化酶并高效表达的基础上，通过对酶的结构解析,利用定点突变技术在酶分子表面远离活性中心且富含Lys残基的固定化部位引入"定向"功能残基-Cys残基；其次制备核壳型纳米磁性载体，并对材料表面进行修饰，得到可与酶分子表面Cys残基以及Lys残基(多点共价固定化)对接的活性基团；最后通过控制反应条件将酶突变体定向固定到磁性纳米载体表面。本研究将为利用蛋白质工程技术设计分子水平高效固定化酶提供理论依据和实践经验，同时促进NAD再生技术的发展，具有广阔的应用前景。

氧化还原酶是所有酶类中最大的一类，具有高度催化专一性，由于其需要价格昂贵的辅酶，所以至今未见有工业化的先例。其中需要利用NAD作为辅酶的氧化还原酶大约为80%，因此如果能够采用绿色技术高效再生NAD，则氧化还原酶的工业化再生瓶颈有望被打破。本研究基于申请者在前期克隆到的高活性NADH氧化酶，该酶可高效催化NADH生成NAD和H2O。本研究对NADH氧化酶进行结构分析，选取表面可突变位点，在不改变或者较少酶催化活性的基础上引入固定化功能残基Cys，同时制备相应的功能载体，实现定向酶固定化，并将固定化酶应用于NAD辅酶再生中，实现氧化还原酶的高效辅酶再生。.本项目首先制备了多种磁性纳米载体，利用水热法和共沉淀法分别制备了Fe3O4@SiO2材料，并对其表面进行了多种基团修饰，获得可供固定化的氨基、环氧基和醛基；同时合成了石墨烯载体，应用于酶的固定化。对固定化载体表征后，对NADH氧化酶的固定化中，获得了高于90%的固定化效率和收率。除了NADH氧化酶，还对其他酶类如脂肪酶，青霉素酰化酶，纤维素酶等酶进行了固定化，固定化的结果表明，制备的载体也是其他酶类较好的固定化载体。.分子建模的结果显示，在酶分子的表面，存在3个富含Lys残基的区域，在这3个区域里，我们选择了8个位点，突变成Cys残基。对突变体的性质分析发现，所有突变体均提高了酶活力，对底物的亲和力有不同程度的提高。其原因还在进一步分析中。.对固定化NADH氧化酶的应用进一步进行验证，在利用固定化醇脱氢酶合成R-扁桃酸的应用中，获得了64%的收率；在和固定化甘油脱氢酶耦合合成1，3-二羟丙酮中，与无NAD再生体系相比，产物收率提高了4倍。.项目成功通过对NADH氧化酶进行突变，制备对应载体，实现定向固定化NADH氧化酶，并应用于辅酶再生中，对于氧化还原酶的工业应用具有推动作用。