Construction Of Two Enzyme-coupled Coenzyme NAD(H) Regeneration Systems And The Applications

Within the coenzyme-dependent reaction system,the coenzyme in situ regeneration system uses a catalytic oxidation/reduction method to achieve a cycle of coenzyme between the oxidized state and the reduced state.Catalytic coenzyme regeneration can significantly reduce the amount of coenzyme input in industrial production and the production costs.In this study,a NADH regeneration system was constructed by coupling alcohol dehydrogenase with glucose dehydrogenase and applied to the synthesis of?S?-4-chloro-3-hydroxybutyrate ethyl ester??S?-CHBE?,a high-value pharmaceutical intermediate.Meanwhile,the NAD⁺regeneration system was constructed by coupling arabitol dehydrogenase and NADH oxidase,and applied to the synthesis of rare xylulose.The research contents are as follows:Construct expression vectors and transformation:the target genes from genomic DNA extracted from Bartonella apis,Bacillus sp.ZJ and Aspergillus nidulans were cloned by PCR.According to their source strains,alcohol dehydrogenase,glucose dehydrogenase and arabitol dehydrogenase were named BaADH,BsGDH and ArDH,respectively.The genes were ligated into the expression plasmid pET-28a?+?and the recombinant plasmids were transformed into the expression host E.coli BL21.Enzyme expression and characterization:the optimum temperature of BaADH and ArDH was 50°C and 40°C,respectively.The two enzymes showed maximum activity in buffers of pH 8.0 and 9.5,respectively.Mn²⁺was found to significantly increase their catalytic activity.BaADH lost 50%of its activity after 35 h of incubation at 30°C,while the half-life of ArDH was 8.3 h.The data of enzymatic reaction rate showed that for BaADH,V_max=190.4 U/mg,K_m=0.11 mM;for ArDH,V_max=9.1 U/mg,K_m=1.2 mM.BaADH and BsGDH were combined to construct NADH reduction system.Operational conditions like temperature,pH,coenzyme concentration and substrate concentration were optimized.The results showed that in the pH 7.0 Tris-HCl buffer,the system reached the maximum conversion?94.9%?after 8 h reaction at 35°C.When 0.5?M of NADH was added,the total turnover number?TTN?reached 5940,which has reached the requirements of industrial applications.ArDH and Nox were combined to construct NAD⁺reduction system and the reaction conditions were optimized.The results showed that after 20 mM arabitol were added to the pH 5.0 citrate buffer,91.5%of arabitol was converted to xylulose within 8 h at 30°C.The highest TTN reached 1020 after 5?M of NAD⁺was added.In this work,an enzyme-coupled regeneration system for NADH and NAD⁺was established.These systems could be used to produce pharmaceutical intermediates and rare sugars,and showed the potential application of coenzyme regeneration system in industrial production.