The Study Of Gene Mining For Aldo-keto Reductases And Reaction Engineering

Herein,three aldo/keto reductases?AKRs?were obtained and used to prepare N-ethyl-methyl-carbamic acid-3-[?1S?-hydroxy-ethyl]-phenyl ester??S?-NEMCA-HEPE?,which is a key intermediate of Rivastigmine for treating Alzheimer's disease?AD?.The reaction rate and conversion of AKR-catalyzed reactions were improved by constructing the recombinant cell and introducing additives to the system.For the asymmetric reduction of N-ethyl-N-methyl carbomyl acetophenone?NEMCA?to prepare?S?-NEMCA-HEPE,we screened the enzymes that could catalyze the reaction in NCBI database.Three AKRs gene?ytbE iolS,yhdN?from the selected Bacillus subtilis 168 genome were obtained and successfully expressed in E.coli BL21?DE3?.The optimal induction temperature was 25?and the final IPTG concentration was 0.1 m M.The crude AKRs can be purified by Ni-NTA metal affinity chromatography.The specific activities of purified ytbE,iol S and yhd N enzymes were0.6 U/mg?protein?,2.4 U/mg?protein?and 0.5 U/mg?protein?,respectively.The studies on the enzymatic properties of AKRs showed that AKRs were suitable to perform good activities in phosphate buffer?pH 6.5-7.0?at 30-40?.Except for Ca²⁺and Mg²⁺can weakly activate the activities of iolS and yhd N,most of the metal ions can inhibit the activities of AKRs in different degrees and the most obvious activities inhibition were caused by Cu²⁺and Fe³⁺.The obtained free AKR enzymes were employed in the reduction reactions of NEMCA.The results revealed that all of AKRs could asymmetric reduce NEMCA to obtain?S?-NEMCA-HEPE with e.e._p of 99.9%.When NAD⁺participated in the cofactor regeneration,the reaction conversion catalyzed by iolS,ytbE and yhdN were10.1%,15.3%and 0.8%,respectively.While the reaction system containing cofactor NADP⁺,the reaction conversion catalyzed by iolS,ytbE and yhdN were 19.2%,18.4%and 5.7%,respectively.Obviously,the catalytic activities of the AKRs were different in presence of different cofactors,and they have a priority to depend on NADP⁺.In all free AKRs-catalyzed reactions,iolS has the highest catalytic activity in presence of NADP⁺,and the reaction conversion was 19.2%in 24 h.For the different catalytic properties of AKR enzymes in the reduction of NEMCA,molecule docking was performed in this study to analyze the interactions between AKRs and NEMCA.The results showed that both the hydrogen bonding and?-?stacked interactions,especially the hydrogen bonding interactions in AKRs-NEMCA complexes played an important role for promoting the carbonyl reduction of NEMCA.To avoid the usage of extra cofactors,the three recombinant whole-cells containing AKR and glucose dehydrogenase?GDH?were constructed and applied in the reduction reactions.The whole cells iolS-GDH,ytbE-GDH and yhd N-GDH can catalyze the reactions with the conversions of 83.3%,80.0%and 78.2%within 1 h,respectively.The whole cell iolS-GDH exhibits the excellent catalytic activity.The reaction tendency catalyzed by three AKR-GDH wholecell was consistented with the catalytic results of free AKRs.But all of the constructed AKR-GDH whole cells exhibited the excellent reaction rates and conversions compared with that of free AKR enzymes,and the high optical pure?S?-NEMCA-HEPE?e.e._p=99.9%?can be achieved in all of whole cell-catalyzed reactions.To further improve the cell-catalyzed reaction rate and conversion,different additives were introduced to the reaction system,such as ionic liquids?ILs?,deep eutectic solvents?DESs?and organic solvents.In particular,the addition of1-butyl-3-methylimidazolium tetrafluoroborate?[BMIm]BF₄,5%?v/v??in the whole cell-catalyzed reaction system can dramatically increase the reaction conversion from83.3%?control group?to 98.3%only in 1 h.Moreover,the reaction time?1 h?was much shorter than 24 h that reported in the literature.In order to explain why the additives can improve the performance of whole cell-catalyzed reaction,the flow cytometry?FCM?and ultraviolet spectrum were used to analyze the permeability of cell membrane affected by different additives.The results of the two methods matched the reaction datas,which can prove that different additives do have different effects on the cell membrane,and the moderate damage of cell membrane is beneficial to the whole cell-catalyzed reaction.This study provided a simple,efficient,environmentally friendly and low-cost method for the preparation of?S?-NEMCA-HEPE with high optical purity,which has the potential of industrial application.