Study On The Synthesis Of 2'-fluoro-2'-deoxyadenosine Catalized By Nucleoside Phosphorylases

Nucleoside analogs have been widely used in cancer and antiviral treatments,but their expensive drug costs have hindered extensive biological research and therapeutic applications.The traditional chemical synthesis method has many steps and takes a long time.It also requires technically difficult glycosyl activation,stereo-and region-selective control of glycoside formation.Therefore,biocatalysis that can effectively synthesize nucleoside analogues under environmentally friendly conditions provides a more attractive alternative way to make the process more efficient and clean.In this paper,thymidine phosphorylase(TP)and purine nucleoside phosphorylase(PNP)were selected as biocatalysts to catalyze the transglycosylation of 2'-fluoro-2'-deoxyuridine and adenine which produces 2'-fluoro-2'-deoxyadenosine.Firstly,according to the spatial structure of the purine nucleoside phosphorylase of E.coli,the active sites on the original sequence of PNP were selected for site-directed saturation mutation.The concentration of the product was detected by high-performance liquid chromatography.The mutant with the best enzyme activity was selected,and the enzyme activity increased by 47.6%compared with the wild-type strain.Secondly,the culture conditions of the mutant strain were explored and the optimized conditions were:inoculated with 2%(v/v)LB activated bacterial solution,added kanamycin to a final concentration of 40 mg/L,and cultured in a shaker at 37?,200 rpm.When the OD600 of Solution reaches 0.5 Abs,add IPTG to a final concentration of 0.9 mM and incubate at 33?,200 rpm shaker for another 12 h.And in the 5 L fermentor,the fermentation test of the engineering bacteria was carried out,and a total of 198 g of wet cells were obtained.Finally,optimization of the transglycosylation reaction of 2'-fluoro-2'-deoxyuridine and adenine catalyzed by the most excellent variant MF13 and TP were explored.The best reaction conditions were:reaction temperature 48?,100mM phosphate buffer solution pH=7.0,2'-fluoro-2'-deoxyuridine 25 mM,adenine 62.5 mM,2 mL TP enzyme solution,2 mL MF13 enzyme solution.The reaction yield can reach 60.2%.On the basis of reaction conditions,the reaction system was enlarged 50 times.After 36 hours of reaction,the concentration of product was 3.85 g/L,and the yield reached 57.4%.