氧化葡萄糖酸杆菌mPQQ-GDH对木质纤维素来源酚醛抑制物耐受性结构基础解析

The production of gluconic acid from lignocellulosic biomass instead of grain has become an important research field. Lignocellulose derived inhibitors have become the bottleneck of the development of gluconic acid industry in biorefinery field. A key enzyme of gluconic acid production in Gluconobacter oxydans, membrane-bound and PQQ-dependent glucose dehydrogenase (mPQQ-GDH), showed a strong tolerance to phenolic aldehyde inhibitors. Structural characteristics of mPQQ-GDH could provide the rational design basis for enhancing the inhibitor tolerance of key industrial enzyme in the biorefinery industry, so structural analysis of mPQQ-GDH is significant and necessary. In this project, the mPQQ-GDH of G. oxydans was overexpressed and purified in Escherichia coli, and the enzymatic kinetics characteristics under stress of phenolic aldehyde inhibitors were studied. After selecting the crystals, the crystal structure of mPQQ-GDH was analyzed by X-ray crystallography. mPQQ-GDH mutants with significant differences in phenolic aldehyde tolerance were obtained through directed evolution technology, and then the key amino acid residues that significantly affect the structural stability of mPQQ-GDH were mined from these mutants according to the crystal structure information of mPQQ-GDH. The mPQQ-GDH mutant with stronger tolerance to phenolic aldehyde inhibitor was screened by rational design based on the structural characteristics of this enzyme. Finally, the structural characteristics of mPQQ-GDH tolerance to phenolic aldehyde inhibitors was clarified by integrating the crystal structure and biochemical information of mPQQ-GDH.

利用木质纤维素生物质替代粮食生产葡萄糖酸成为重要的研究领域，而抑制物成为阻碍葡萄糖酸生物炼制产业发展的瓶颈。氧化葡萄糖酸杆菌中葡萄糖酸生产关键酶-膜结合PQQ依赖型葡萄糖脱氢酶（mPQQ-GDH）对酚醛抑制物有较强的耐受性，其结构特性将为增强生物炼制产业关键工业酶的抑制物耐受性提供理性设计基础，对mPQQ-GDH的结构解析具有重要意义。本项目将mPQQ-GDH在大肠杆菌中过量表达、纯化，对酚醛抑制物胁迫下的酶学特性进行研究。筛选晶体后，通过X射线晶体学技术解析其晶体结构。通过定向进化获得酚醛耐受性发生显著差异的突变体，依据mPQQ-GDH晶体结构信息，从中挖掘显著影响mPQQ-GDH结构稳定性的关键氨基酸残基。结合酶的结构特征进行理性设计，筛选得到对酚醛抑制物耐受性更强的mPQQ-GDH突变体。整合mPQQ-GDH的晶体结构与生化研究信息，阐明mPQQ-GDH对酚醛抑制物耐受性的结构特征。

木质纤维素生物质是一种来源广泛、储量巨大的可再生有机资源，当前生物质资源化利用率低，不仅造成了巨大的资源浪费还产生了严重的环境问题。使用木质纤维素原料代替传统粮食原料生产生物基化学品具有重要意义，而木质纤维素来源抑制物成为阻碍生物炼制产业发展的瓶颈。Gluconobacter. oxydans中合成葡萄糖酸的关键酶（膜结合的PQQ依赖型的葡萄糖脱氢酶，mPQQ-GDH）在酚醛抑制物存在下依然保持高催化活性，表现出对酚醛抑制物的高耐受性，从而使得G. oxydans能够在高浓度玉米秸秆水解液中高效生产葡萄糖酸，其结构特性将为增强生物炼制产业关键工业酶的抑制物耐受性提供理性设计基础，因此对mPQQ-GDH的研究具有重要意义。本项目通过密码子优化、表达载体优化成功将mPQQ-GDH表达载体转入大肠杆菌中进行异源表达得到可溶性的重组膜蛋白。mPQQ-GDH的酶学性质、反应动力学、抑制物耐受性研究结果表明mPQQ-GDH的最适pH为6.4，最适反应温度为25℃。mPQQ-GDH的催化活性在不同来源葡萄糖脱氢酶中处于较高的水平。mPQQ-GDH对木质纤维素来源的酚醛抑制物比G. oxydans内另一个葡萄糖脱氢酶（NADP-GDH）高40%。在抑制物胁迫下的酶动力学参数结果为Vmax减小，Km基本保持不变，表现为可逆的非竞争性抑制，即抑制物不与活性位点结合，不影响Km，而是可逆的结合到酶的其它位点上从而使得酶的三维结构改变但并不会破坏活性位点。通过对其结构进行预测解析，结合定点突变，挖掘出多个影响mPQQ-GDH抑制物耐受性及热稳定性的关键氨基酸残基，其中W408A突变体表现出更强的抑制物耐受性以及热稳定性。综上，本项目从一定程度上解析了mPQQ-GDH酚醛抑制物耐受性的结构特征。关键氨基酸残基的挖掘有助于指导酶的理性设计以获得抑制物耐受性更强的mPQQ-GDH，为富含抑制物的高浓度木质纤维素葡萄糖酸体外催化生产提供物质基础，并为生物炼制中更多关键酶的抑制物耐受性定向改造提供理性设计依据。