Study On The Technology Of Enzymatic Synthesis Of Thymidine And Cytidine

In terms of the technologies of enzymatic synthesis of thymidine and cytidine with nucleoside phosphorylase of E.coli,the optimized conditions have been obtained by study the conditions in biotransformation reaction.The results of the study can provide certain theoretical foundation for the industrialized mass production.Therefore,it will have great practical significance and socio-economic value.Firstly,The paper studied the Cell-free synthesis of thymidine and cytidine.nucleotides were synthesized with cheap nucleotides and bases as substrates by the nucleoside phosphorylase of E.coli.Then,the cell culture time,the reaction time,pH and temperature,which affected the transformation ratio of the enzymatic synthesis were studied.The results of three-factor-three-level orthogonal tests showed that 20.0ml reaction solution（cytidine 30.0 mmol/L,thymine 90.0 mmol/L,KOH-KH₂PO₄（pH 7.05）,1.0g wet cells of E.coli）reacted constantly in 60℃for five hours, the transformation ratio of thymidine can be up to 27.1%.Experiments also showed that the reaction solution（pH 7.05）,reacted constantly in 60℃for four hours,the maximum transformation ratio up to 12.3%was achieved.Then the paper studied the synthesis of thymidine and cytidine with immobilized cells,immobilization carrier and preparation technology were researched specifically.In order to obtain the optimal conditions of immobilization,the formula ratio,the methods of calcify and crosslinking and the storage methods were studied.The optimal condition of alginate microsphere was:sodium alginate 2.0%,MgO 1.4%,blending E.coli as volume ratio of 10:1,using 2.0%CaCl₂ as crosslinking agent,standing at room temperature for four hours to calcify.Cryopreservation is the best storing method of sodium alginate.When polyvinyl alcohol 10.0%, sodium alginate 0.8%,blending E.coli as volume ratio of 3:1,using mass fraction as 4.0%CaCl₂-saturated solution of boric acid as crosslinking agent,standing at room temperature for four hours to crosslinking that can get the optimal preparation conditions of polyvinyl alcohol microsphere.