Cloning, Expression And Application Reach Of N-deoxyribosyltransferase-Ⅱ

It has been proved by many study that nucleoside analogues have potential therapeutic effect in cancer or viral infection.It became a hot point in recent years.Nucleoside synthesise and transformation only need one step by N-deoxyribosyltransferase,while as reported before,there must need purine nucleoside phosphorylase and pyrimidine-nucleoside phosphorylase.Therefore engineering bacteria with N-deoxyribosyltransferase-Ⅱwas studied in this paper on nucleoside transformation.The N-deoxyribosyltransferase-Ⅱgene was PCR amplification from Lactobacillus helveticus using primer designed.The gene was connected with pMD18-T,and then identified by sequencing and BLAST.If the result was correct,the enzyme gene was cut from pMD18-T and connected with pET-28a,transformed into Escherichia coli BL21(DE3).In the end,we got a engineering bacteria contain gene N-deoxyribosyltransferase-II.The engineering bacteria was induced in different temperature,induction time and IPTG concertration in order to find the optimum expression condition.The engineering bacteria expressed a large number of aimed protein which was mostly soluble protein for 4h induction at 37℃induced with 0.1mmol/L IPTG.The supernate of bacteria extractation after induction was used as enzyme.Enzyme activity of engineering bacteria was 22 times higher than that of orgin Lactobacillus helveticus.Optimum conditions for synthesizing deoxycytidine and deoxyadenosine from thymidine were studied,such as temperature,reaction time,pH,substrate concentration and so on.The best condition of deoxycytidine synthesis was as following,5mmol/L substrate concentration,pH7.1 Tris-HCl and 200μL enzyme solution,60℃for 2h.The highest conversion rate was 59.19%.The best condition of deoxyadenosine synthesis was 5mmol/L substrate concentration,pH7.1 Tris-HCl and 200μL enzyme,60℃for 9h,which the highest conversion rate was 82.01%.In addition,some transition metal ions were found inhibition reactions.5-Aza-deoxycytidine(5-Aza-CdR),an antineoplastic agents drug,was enzymatically synthesized under the following condition,2mmol/L substrate concentration,pH7.1 Tris-HCl and 200μL enzymesolution,60℃for 2h,which the highest conversion rate was 61%.