Construction And Application Of Enzymatic Regeneration Systems Of Oxidized Nicotinamide Cofactors

Oxidoreductases are an important type of industrial biocatalysts,which emerge as a novel tool in the fields of organic synthesis and pharmaceuticals.Cofactors are usually required in oxidoreductases-catalyzed reactions.In addition,most of oxidoreductases are nicotinamide cofactor（NAD（P）（H））-dependent.From the economical point of view,it is impossible to use stoichiometric natural nicotinamide cofactors for the reactions,because they are costly.Therefore,the construction of efficient cofactor regeneration systems is important for the applications of oxidoreductases in organic synthesis.Compared to the NAD（P）H regeneration,the recycling of their oxidized forms NAD（P）⁺appears to be more difficult.And less attention was paid on the NAD（P）⁺recycling than the NAD（P）H regeneration.There are limited examples of preparative dehydrogenase（DH）-mediated oxidations based on the NAD（P）⁺recycling.In this thesis,heme protein-catalyzed regeneration of oxidative nicotinamide cofactors was studied based on their catalytic promiscuity;and dual and multi-enzyme systems were constructed for catalytic oxidations.The main results are summarized as follows.（1）Hemoglobin（Hb）-catalyzed regeneration of NAD（P）⁺.It was found that Hb was capable of oxidizing NAD（P）H with H₂O₂,affording NAD（P）⁺.The optimum reaction temperature and pH for this reaction were 30°C and 8.0,respectively.This regeneration method coupled with horse liver alcohol DH（HLADH）was used for the oxidation of furfural.The yield of 2-furoic acid was 96%after 60 h,with a total turnover number（TTN）for cofactor of approximately 960.This dual-enzyme system was applied for the oxidation of different aryl alcohols and aldehydes,and the product yields were>81%.In addition,the enzymatic regeneration method was integrated with L-glutamate DH（L-glu DH）as well as L-lactate DH（L-lac DH）for the oxidation of L-glutamate and L-lactate,respectively.α-Ketoglutarate and pyruvate were obtained in the yields of 97%and 81%,respectively.It demonstrates the good enzyme-compatibility of the regeneration method.（2）Myoglobin（Mb）-catalyzed regeneration of NAD（P）⁺and its mechanism.To further improve the cofactor regeneration efficiency,other heme proteins（cytochrome C,horseradish peroxidase（HRP）and Mb）were explored for the oxidative regeneration of NAD（P）⁺.Compared to Hb,Mb with a simpler structure exhibited higher catalytic efficiency.In addition,Mb is capable of effective regeneration of the natural cofactors NAD（P）⁺as well as the synthetic biomimetic 1-benzyl nicotinamide（BNA⁺）,indicating that Mb has broad substrate scope in the regeneration of oxidized cofactors.The Mb-catalyzed NAD（P）⁺regeneration efficiencies were greatly improved in the presence of some phenols（as mediators）,and the optimal mediator was scopoletin.The Mb-catalyzed NAD⁺regeneration method was applied for glucose DH（GDH）-catalyzed oxidation of glucose;gluconic acid was synthesized with the yield up to 97%,with the cofactor TTN of around 44 000.Also,Mb coupled with other DHs including L-glu DH and L-lac DH was applied for catalytic oxidations,affording the desirable products with the yields of>95%.Besides,HLADH integrated with Mb-catalyzed regeneration of BNA⁺enabled furfuryl alcohol to be oxidized to 2-furoic acid,with the yield of 97%and the cofactor TTN of 190.Based on the study on the catalytic mechanism,Mb was rapidly transformed into a plausible heme-bound peroxide(Fe^IIIOOH Por)in the presence of H₂O₂,and the O-O bond of Fe^IIIOOH Por could be cleaved both heterolytically and homolytically to form extremely active ferryl species Mb-I and Mb-II,which were reduced sequentially into the ferric state through the one-electron oxidation of the mediator scopoletin.The highly catalytically active phenoxy radical are formed.Then,the phenoxy radical rapidly oxidized NADH into NAD^·,followed by conversion into the oxidized cofactor NAD⁺in the presence of O₂.（3）Application of the regeneration system in ADH-catalyzed synthesis of furan carboxylic acids.The substrate scope of three ADHs including Streptomyces coelicolor carbonyl reductase（Sc CR）,Synechocystis sp.ADH（SADH）and HLADH was investigated.It was found that the three enzymes were able to oxidize aldehydes.Sc CR and SADH were combined with the enzymatic regeneration system for the oxidation of 5-hydroxymethylfurfural（HMF）and 2,5-formylfuran（DFF）,respectively.The desired products5-hydroxymethyl-2-furancarboxylic acid（HMFCA）and 5-formyl-2-furancarboxylic acid（FFCA）were obtained with the yields of 96%and 98%,respectively.In addition,HMFCA was obtained by using Sc CR on a laboratory-preparative scale（50 m L）with a 92%isolated yield and 95%purity.HLADH proved to be an enzyme with broad substrate specificity,and it enabled DFF to be oxidized to 2,5-furandicarboxylic acid（FDCA）with BNA⁺.（4）Application of the regeneration system in multi-enzymatic synthesis of furan carboxylic acids.HRP was capable of recycling NAD⁺as well as of activating galactose oxidase（GO）.So HRP was coupled with GO and DHs for the oxidation of HMF to furan carboxylic acids.HMF was oxidized to DFF by GO,along with the formation of byproduct H₂O₂;the latter was used by HRP to oxidize NADH to NAD⁺;finally,the resultant NAD⁺was used by DHs to convert DFF to FFCA or FDCA.The combination of Dactylium dendroides GO,ADHs and HRP was used for the oxidation of HMF.FFCA was obtained with the yields of up to 98%and cofactor TTN of approximately 10 000,while the FDCA yield was up to95%.To improve the catalytic efficiencies of the multi-enzyme system as well as reducing enzyme amounts,the enzyme screening was re-conducted.It was found that the multi-enzyme system composed of GO M_3-5（from Fusarium sp.）,Geotrichum candidum aldehyde DH（Gc ALDH）and HRP was capable of efficiently oxidizing HMF.After 48 h,FFCA was obtained in the yield of>99%and the cofactor of around 5 000.The synthesis of FFCA was scaled up（50 m L）,and this substance was obtained with an isolated yield of 89%and the purity of 98%.In addition,the one-pot two steps multi-enzyme cascade system composed of GO,Gc ALDH,HRP and Pseudomonas aeruginosa for the synthesis of high-valued FDCA from HMF.After 72 h,FDCA was obtained in the yield of 75%.The synthesis of FDCA was scaled up（50 m L）,and it was obtained with an isolated yield of 72%and the purity of 93%.In summary,heme protein-catalyzed regeneration of oxidized nicotinamide cofactors was studied based on their catalytic promiscuity;and dual and multi-enzyme systems were constructed for catalytic oxidations.This study not only establishes new methods for the regeneration of oxidized nicotinamide cofactors,but also provides a new pathway for the clean and green synthesis of furan carboxylic acids.