Preparation And Characterization Of NADP~+ Dependent Formate Dehydrogenase

Oxidoreductases are extensively used in the fields of pharmaceuticals,fine chemical synthesis and also in the production of chiral materials.The completion of most of the reactions requires nicotinamide adenine dinucleotide(NADH)or its phosphorylated form,niacinamide coenzyme(NADPH),while whose disadvantages including high price,low efficiency in application and re-use.Hence,it is necessary to construct a suitable coenzyme regeneration system to solve such problems.The formate dehydrogenase(FDH)regeneration system shows the highest economic efficiency in all the constructed enzyme-coupled coenzyme regeneration systems,of which NADP~+-dependent formate dehydrogenase is the key enzyme in the oxidized and reduced coenzyme systems.Therefore,it is of great significance to develop methods to obtain NADP~+-dependent formate dehydrogenase.In this paper,the NADP~+-dependent formate dehydrogenase was obtained in two routes.The first approach to obtain NADP~+-dependent formate dehydrogenase is genetic engineering.Burkholderia stabilis 15516 formate dehydrogenase(BCC-FDH)encoding gene(BCC-fdh)was amplified by PCR from B.stabilis ACF35003.1 genomic DNA.The gene is constituted by 1161 bp nucleotides,which encode 386 amino acids.The recombinant vector pET-17b-FDH was constructed with pET-17b as expression vector and transformed into E.coli BL21(DE3).A series of gradient experiments were conducted to analyse the optimal conditions of enzyme production as well as the enzymatic properties.The results showed that the optimum induction time was 10 hrs after inoculation while the best induction time was 28hrs,with the optimal concentration of IPTG at 0.5 mM.Furthermore,the optimum temperature and pH of reaction was 30°C and 6.0,respectively.The enzyme has a wide optimum pH(pH 6.0-9.0)and stability overa broad pH range.At the temperature range of25-30°C,the recombinant enzyme had little effect on the specific activity of NAD~+and on NADP~+when the temperature was increased to the range of 30-37°C,hence thermal stability needs to be improved upon.Another method of obtaining NADP~+-dependent formate dehydrogenase is to collect wild-type.This method involves collecting of soil samples surrounding the plant roots,in which the Burkholderia cepacia and related species were directly screened using TB-T and PCAT plates.Based on colony morphology,KOH solution method and the Gram staining method,563 suspected isolates were screened.However,due to environmental degradation and bad soil quality,the traditional microbial separation method is characterized by high repeatability,heavy workload with low efficiency and long cyclability,the work is still in progress.In view of the above two methods,this study established different ways to screening NADP~+-dependent formate dehydrogenase andgiven the large number of wild strains and the high repetition rate of strains,the use of trace color screening can quickly and accurately detect samples.For the recombinant bacteria,the crude enzyme solution was directly added to the enzyme activity test system,and the OD600 was measured by a UV-visible spectrophotometer to calculate the enzyme activity.