| Formate dehydrogenase（FDH,EC 1.2.1.2）is commonly used in the recycling of NADH,a reduced coenzyme,in the production of biotransformed amino acids,it can transfer hydrogen with formate and oxidized coenzyme NAD+as substrate,and the by-product CO2is easily excreted,which has little effect on p H of the conversion system,therefore,it is considered to be one of the best enzymes for NADH regeneration in the synthesis of optically active compounds,and is widely used in food,pharmaceutical and chemical industries.However,the low activity and low catalytic efficiency of wild-type formate dehydrogenase are common problems,and the high flexibility of the protein structure of wild-type formate dehydrogenase determines that wild-type formate dehydrogenase is easy to deactivate at the temperature needed for production,result in low product conversion rate,affect the industrial production of products.Therefore,the improvement of the activity and thermal stability of wild-type formate dehydrogenase by molecular biology and bioinformatics has a broad application prospect for the production and application of chiral amino acids such as L-norvaline.L-norvaline is an endogenous metabolite that is used in the synthesis of nutrients and drugs.For example,l-norvaline is used as a key intermediate in the synthesis of Perindopril,a common antihypertensive drug.Perindopril is considered to be one of the most effective,safe and widely used drugs for the treatment of hypertension and cardiovascular diseases because of its long-lasting inhibition of angiotensin-converting enzyme（ACE）,which does not contain sulfhydryl groups,for the treatment of hypertension and heart failure.In this study,by modeling the protein structure of formate dehydrogenase（FDH）and using biological software to predict the mutation point of the FDH protein model,according to the predicted results,molecular modification was carried out and the forward mutants were screened out for further thermal stability modification.The modified mutant formate dehydrogenase was used in L-norvaline conversion system,and the synthesis efficiency of l-norvaline was improved:（1）FDH from Candida boidinii was used as a template of HOTSPOT WIZARD v3.1webserver for predicting mutational hotspots to obtain a variant with improved catalytic activity and bioconversion.The three-dimensional protein structure was predicted and simulated.Two mutants,P68G and Q197K,were constructed.The specific enzyme activities were increased by 11.4%and33%,respectively,compared with the wild type.（2）On the basis of single point mutation,a cysteine mutation was introduced to form a disulfide bond with C262,and the finally mutant strain Cb FDHQ197K/I239Cwith significantly improved thermal stability,31%higher specific enzyme activity than wild type was obtained,the mutated gene is named cbfdh2.（3）On the basis of the high-efficient synthesis of l-norvaline plasmid in the early stage of the laboratory,the recombinant plasmid p ET28a-Rtdaao S-Bpcat-Cbfdh2-Bcldh was constructed and introduced into E.coli BL21 to obtain the recombinant expression strain E.coli BL21/L-nor2 to construct a multi-enzyme cascade system.The substrate concentration was 300 m M.The transformation time of recombinant E.coli BL21/L-nor2 was 12 h which was 4 h faster than that of recombinant E.coli BL21/L-nor,and its transformation rate was 1.33 times that of E.coli BL21/L-nor.（4）E.Coli BL21/l-nor2 continuous batch transformation test showed that the reutilization rate of the whole cell system was up to 5 times,and the transformation efficiency was good in the reutilization system.In the scale-up experiment of 5 L fermenter,L-norvaline was produced continuously,and the conversion rate was 99.9%. |
