生物催化5-羟甲基糠醛(HMF)选择性氧化反应及其机理

With depletion of fossil resources and increasing concern of biorefinery, the green production of bio-based chemicals has emerged as an interesting research field. 5-Hydroxymethylfurfural (HMF), an important bio-based platform chemical, could be readily transformed into various value-added products. However, there are limited reports about biotransformation of HMF, and the enzymes which are capable of catalyzing the efficient conversion of HMF are seldom. In this project, we’d like to use the strategy of solvent engineering to relieve the inhibitory effects of the substrate and products against the microorganisms. A comparative study of whole cells-catalyzed oxidation of HMF in different biphasic systems composed of various nonaqueous solvents and buffer will be conducted to elucidate the influential law of nonaqueous solvents on the biocatalytic performance. An efficient biocatalytic approach to the oxidized derivatives of HMF will be developed. Then, the key enzyme that is responsible for HMF oxidation will be mined, and its catalytic performances in the HMF oxidation and its structures in the nonaqueous solvents will be investigated, to understand the molecular basis of the nonaqueous solvents’ effect on the enzyme reaction. Finally, the spatial structures of the key enzyme and enzyme-substrate complex will be resolved to reveal the key amino acid residues in the substrate-binding and catalytic sites of the enzyme, thus uncovering the reaction mechanism. The present study will provide the theoretical guidance for the rational design of the new enzymes.

近年来，随着石油资源的日益枯竭和生物精炼概念的兴起，生物基化学品的绿色加工与制造已成为了一个研究热点。5-羟甲基糠醛（HMF）作为一个重要的生物基平台化合物，可转化为各种高附加值中间体。然而，国内外鲜有关于HMF生物转化的报道，且能高效催化HMF转化的酶仍很少。本项目以自行筛选得到的微生物为研究对象，借助介质工程手段解除底物及产物对微生物的抑制作用，对比研究不同非水介质/水双相体系中全细胞催化HMF氧化反应性能，阐明非水介质对全细胞催化HMF氧化反应的影响规律，建立高效合成HMF氧化衍生物的生物催化途径；在此基础上，挖掘催化HMF转化的关键酶，研究非水介质中该酶催化HMF反应性能及酶结构，揭示非水介质对该酶催化HMF氧化反应影响的分子基础；进而解析该关键酶、酶-底物复合物的三维结构，探索酶的底物键合及催化位点的关键氨基酸残基，阐明其催化机制，为后续酶分子的理性设计和分子改造提供理论指导。

5-羟甲基糠醛(HMF)是一个重要的生物基平台化合物，可选择性氧化为高附加值的5-羟甲基糠酸（HMFCA）和2,5-呋喃二羧酸（FDCA）。本项目从土壤中成功筛选到一株高HMF耐受性的菌株睾丸酮丛毛单胞菌(Comamonas testosteroni) SC1588，系统地研究了其催化HMF选择性氧化合成HMFCA反应特性。并对该菌进行基因组测序，发掘出5个与HMF氧化相关的醛脱氢酶，阐明了其催化HMF氧化机制。借助基因工程手段，在大肠杆菌中进行异源表达、分离纯化和酶学性质研究；同时探索了重组大肠杆菌催化HMF氧化性能，结果表明过量表达醛脱氢酶的重组菌能够更高效地催化HMF及糠醛等的选择性氧化合成相应的呋喃羧酸。对过量表达醛脱氢酶的大肠杆菌进行辅因子工程化改造，通过共表达NAD(P)H氧化酶（NOX）促进胞内NAD+再生，从而极大地增强了重组菌催化生物基呋喃氧化性能，建立高效合成呋喃羧酸的生物催化体系。E. coli_CtVDH1_NOX能在9 h内将250 mM HMF转化为HMFCA，时空产率达3.7 g/L h；E. coli_CtVDH2_NOX合成5-甲氧甲基糠酸的时空产率达5.6 g/L h。将HmfH引入上述重组菌，构建了一个高效合成FDCA的生物催化剂，其合成FDCA的时空产率达0.4 g/L h，产率约为92%。本研究可望为生物催化呋喃羧酸合成的大规模应用奠定基础。此外，我们还探索了血红蛋白和肌红蛋白的催化多功能性，并基于该催化多功能性构建了全新的NAD(P)+原位再生体系，用于醇脱氢酶催化氧化。以葡萄糖脱氢酶催化葡萄糖氧化为模型反应，当葡萄糖浓度达500 mM，目标产物收率达97%，辅因子总转化数（TTN）高达50 000。该NAD(P)+再生体系有望替代NOX用于脱氢酶催化氧化。