Study On Synthesis Of Ribavirin Using Enzymic Method

Ribavirin is a broad spectrum antiviral drugs which could be synthesized by ribose-1-phosphate and 1,2,4-triazole-3-carboxamide(TCA)catalyzed by purine nucleoside phosphorylase.Ribavirin presents an extensive inhibitory effect against a variety of DNA and RNA viruses and because it hardly produces drug resistance and has many acting sites,high curative effect,low toxicity and low side effect,now it has been used in the treatment of various viruses.In previous study,a trimeric purine nucleoside phosphorylase(PNPase)with high catalytic efficiency has been screened.Its inducible expression plasmid pQE30-pupG and a constitutive expression plasmid entitled pQF-pupG have been constructed.In the present dissertation,the two plasmids were transformed into 4 strains of Escherichia coli XL1-Blue,BL21(DE3),DH5a and K27,and thus 8 engineered strains were obtained.The bacterial cells were collected from shake flask fermentation broth,and ribavirin was produced by whole-cell catalysis.The results showed that E.coli XL1-Blue and BL21were proved to be more catalytic activity than DH5a and K27 no matter what it was inducible or constitutive.Moreover,the inducible system of E.coli BL21 by adding lactose in the medium at the beginning of fermentation was a useful method,which produced more bacterial biomass than by the IPTG,while possessing the same catalytic activity.Because lactose inducible system only needed the simple operation and low cost,it has important value for industrial application.With lactose induced E.coli BL21 cells 30 g/L(wet weight)as catalyst,100 mmol/L guanosine and 100 mmol/L TCA as substrates in phosphate buffer solution(pH 7.6),17.6 g/L ribavirin was produced after reaction of 20 h at 55℃ and the substrate conversion ratio reached 72.1%.In order to obtain more enzymic activity,an inducible expression plasmid pET-His-pupG(pET plasmids is the most powerful expressed system of recombinant proteins in Escherichia coli)has been constructed and transformed into Escerichia coli BL21(DE3)to obtain a strain BL21(pET-His-pupG).The bacterial cells were collected from shake flask fermentation broth,and ribavirin was produced by whole-cell catalysis.E.coli BL21 by using lactose as inducer could produce more bacterial biomass than that induced by the structural analogue IPTG,and possessed the same catalytic activity.With lactose induced E.coli BL21 cells 30 g/L(wet weight)as catalyst,100 mmol/L guanosine and 100 mmol/L TCA as substrates in phosphate buffer solution(pH 7.6),19.5 g/L ribavirin was produced after reaction of 20 h at 55℃ and the substrate conversion ratio reached 79.9%.Research indicated that when free cells were used for ribavirin synthsis,they were easily cracked and difficult to reuse which added the cost of producing cells.When a recombinant Escherichia coli BL21(pET-His-pupG)strain which could highly express PNPase was immobilized its catalyzing stability was enhanced remarkably and it could be used repeatedly.Comparing different entrapment agent,the gelatin as the best entrapment agent was determined.By optimizing the gelatin immobilized cell catalytic condition,the results showed that the optimal temperature was 55℃,the optimal entrapped cell concentration was 200 g/L,the optimal immobilized cell dosage was 20 g/L(convert into wet cell weight)and reaction period was 20 h.For the biosynthesis of ribavirin by immobilized cell,100 mmol/L guanosine and 100 mmol/L TCA as substrates in phosphate buffer solution(pH 7.6),after 8 batches of continuous reaction,basically no enzyme activity was lost and the highest conversion ratio could reach 83.0%only 20 hour under the optimal condition.In this dissertation,according to the characteristic of the transformation solution of ribavirin,appropriate extraction and purification process were determined.The decolorizing conditions such as the amount of activated carbon,adsorption time and temperature which effected decoloration of solution were mainly studied.Through determining color removal rate and the ribavirin recovery rate,the optimal decolorizing conditions were determined as follows:amount of activated carbon 1%,adsorption time 20 min and temperature 60 ℃.After decolored solution was handled by concentration,crystallization and recrystallization,the purity of ribavirin could reach 99.1%and total yield of the extraction process was 45.2%.