Study On The Synthesis Of Ribavirin By Purine Nucleoside Phosphorylase

Ribavirin is a nucleoside drug with broad spectrum antiviral,which is widely used in the treatment of a variety of viral diseases due to its characteristics of high efficacy,low toxicity and low resistance to drugs.Enzymatic synthesis of ribavirin by using purine nucleoside phosphorylase to catalyze the reaction of purine nucleosides and TCA,which has great industrial prospects.In this paper,firstly,the culture conditions of Brevibacterium acetylicum that can express PNPase were optimized,and the optimal peptone was determined.At the same time,response surface method was used to increase the bacterial concentration to 16.8 g/L on the premise of ensuring the conversion rate,which increased by 25.4%,and the price was reduced by 28.3%.The gene sequences of three strains of Bacillus subtilis,Bacillus halodurans and Brevibacterium acetylicum were linked with pET20b vector to construct recombinant plasmids,and the enzymes BsPNP,BhPNP and BaPNP were efficiently expressed in E.coli BL21(DE3).By optimizing the expression conditions of the three recombinant bacteria,the expression of the target protein accounted for more than 50%of the total protein.The target protein was purified by nickel affinity chromatography and confirmed by SDS-PAGE.Through the three enzymatic properties of protease to explore,to determine the role of the above three protease optimum temperature is 65 ℃,75 ℃ and 65 ℃,the optimum effect of pH is 7.3,7.0 and 8.0,and determine their under the condition of pH6.0-11.0 have good pH stability,and the trend of temperature stability is basically the same.Among them,BhPNP has the best temperature stability and enzyme activity after incubation for 1 h under 65 ℃ are still about 90%,by measuring the catalytic kinetic parameters found BhPNP highest ability.In addition,all three enzymes follow the michaelis equation.Ribavirin was synthesized by Brevibacterium acetylicum and recombinant bacteria,and the conversion efficiency of the recombinant bacteria was much higher than that of Brevibacterium acetylicum.By optimizing the synthesis process,the reaction time was shortened from 10 h to 2 h,the amount of bacteria was reduced by 10 times,and the substrate concentration was increased from 100 mmol/L to 400 mmol/L.