Clone,Expression And Application Of Nucleoside Phosphorylase

Nucleosides and derivates are the important source of antiviral or anticancer medicines. Many nucleoside drugs such as zidovudine, ribavirin, lamivudine, capecitabine are widely used in clinical practice. Nucleosides and derivates were commonly chemical synthesized, resulting in serious pollution to the environment for large amounts of toxic and hazardous reagents used. For mildness reaction conditions, simple reaction process and high conversion yield, enzymatic synthesis of nucleosides with nucleoside phosphorylase became one of hot research in recent year.In this study, we first construct three kinds of single recombinant strain which express nucleoside phosphorylase:E.coli BL21 (DE3) (pET-11a-deoD) (1), E.coli BL21 (DE3) (pET-11a-udp) (2), and E.coli BL21 (DE3) (pET-11a-deoA) (3). Also dual-expression bacteria was first time contructed (double recombinant plasmids of DUD(4) and DAD(5), tandem recombinant plasmid of TDU(6) and TDA(7)). All the strains shown good stability and had high application value.In this study, genes coding purine nucleoside phosphorylase, uridine phosphorylase and thymidine phosphorylase from E. coli K-12 were cloned into the pET vector and transformed into E. coli BL21 (DE3). Thus 1,2,3 was constructed. All the recombinant bacteria could express a large number of soluble protein induced with IPTG for 3h. Activity of PNPase measured in intact cells was 116U/mg wet cells, UPase's activity was 3532U/mg wet cells and TPase's activity was 3550U/mg wet cells, which was respectively 8.4times,4.8 times and 7.1 times higher than its in host strain. All the activities were much higher than those in the common wild bacteria. If continuously inoculated on non-antibiotics LB agar slant for 15 times, more than 90% recombinant bacteria hosted plasmid.It was found that lactose instead of IPTG can induce the recombinant to express a large amount of enzyme protein. The induction effect of 8mmol/L added into LB medium was similar with that of IPTG. In the process of bacterial culture, the lactose was not only used as inducer, but also its degradation of glucose and galactose could be used as carbon source, which had certain role in promotion the growth of bacteria.As many Nucleosides and Derivatives were synthesized by the conversion between pyrimidine and purine nucleosides catalyzed by 1 and 2 or 1 and 3. Therefore, in this study purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase should be co-expressed in one recombinant bacteria (double recombinant plasmids of DUD(4) and DAD(5), tandem recombinant plasmid of TDU(6) and TDA(7)). The conversion between purine and pyrimidine nucleoside could be achieved with single recombinant bacteria.Four recombinant bacteria were able to express a large amount of the corresponding two kinds of enzyme protein. All the activities in four recombinant bacteria increased significantly. If continuously inoculated on non-antibiotics LB agar slant for 15 times, more than 85% recombinant bacteria hosted plasmid.Recombinant bacteria above-mentioned could be used to enzymatic synthesis of nucleosides efficiently.Highest conversion yield could achieve in 1 or 2h if recombinant strain 1,2,3 was used to synthesize nucleosides. However, in the synthesis reaction 2,3 participated in, the novel product synthesized would be resynthesized into ribose(deoxyribose)-1-phosphate as reaction time went by. While in the synthesis reaction 1 participated in, the novel product synthesized was very stable. Nucleoside could be synthesized between 40-65℃. The suitable reaction pH was 7.0～7.5. For the amount of target enzyme protein expressed in recombinant strain was very high, little biomass was used to obtain higher conversion yield. Cytosine and analogs could not be synthesized with nucleoside phosphorylase, similar with report in literature. If guanine was used as ribose(deoxyribose) acceptor, the conversion yield was very low in the reaction.In order to study two-enzyme reaction, Ribavirin and deoxyribavirin were synthesized. Good conversion yield could obtain if the enzyme reaction condition was as follows,50℃, pH 7.0 phosphate buffer,0.4%(0.2% individual) biomass, reaction 2h. Phosphate was important to the enzymatic synthesis of nucleoside with NPase. For guanine was difficult to dissolve in water, if it was used as ribose(deoxyribose) acceptor, low yield of guanosine and deoxyguanosine was obtained. However, just as the feature of guanine difficult to dissolve in water, if guanosine was used as ribose donor, high yield of other nucleosides could be achieved. When purine nucleosides were transformed into pyrimidine nucleosides, the yield was significantly lower that that in the reaction of pyrmidine nucleosides to purine nucleoside.In order to study the reaction DAD, DUD, TDU, and TDA participated in,2,6-diamino purine nucleoside (DAPR) and 2,6-diamino purine deoxynucleoside (DAPdR) were enzymatic synthesized. The reaction conditions of these four recombinants were similar. The optimum reaction conditions were as follows,50mmol/L phosphate buffer, pH 7.5, final substrate concentration 30mmol/L,0.5% wet intact cells,50℃,2h.In synthesis of other nucleoside with co-expression recombinant bacteria, the yield was very similar to the reaction two recombinant bacteria participated in. Guanine could not be transformed into related nucleoside. Arabinoside uracil could not transformed int arabinoside adenine for arabinoside nucleoside was not substrate of NPase form E. coli.