| Cytidine triphosphate(CTP)is an important intermediates of nucleic acid drugs and pharmaceutical,but its application in clinical is limited because of the high price.It is necessary to simply the production process and increase yield due to the high production costs of CTP.So,in this study,with CMP as substrate,the biological synthesis of CTP was investigated by utilizing free beer yeast and immobilized yeasts that immobilized by SA-NaCS/PDMDAAC microcapsules.Through this way,the conversion rate and concentration of CTP were improved.Firstly,the microcapsules was prepared by combining the polycationic Poly diallyl dimethyl ammonium chloride(PDMDAAC)and two-component polyanionic system which constructed by cellulose sulfate(NaCS)and sodium alginate(SA).The NaCS was prepared with cotton as cellulose.The effect on the yield and viscosity of NaCS of the Various factors(volume ratio of concentrated sulfuric acid and n-propanol,reaction time,ratio of solid to liquid and temperature)were studied,then the optimum prepared condition of NaCS was obtained:reaction mixture of concentrated sulfuric acid and n-propanol is 1:1,reaction time 60 min,ratio of solid to liquid is 1:20,reaction temperature 2℃.In this condition,the obtained NaCS with degree of substitution 0.38 and kinematic viscosity 38mPa·s,the yield is 90%.Subsequently,the concentration of polyanion and polycation,reaction time and other factors were investigated in order to study the diameter,mechanical strength and capsule thickness of microcapsules,thus the optimal condition for the preparation of microcapsules was determined:sodium alginate 1%,NaCS solution 4%,PDMDAAC solution 5%,with reaction 40min at room temperature.Then studying the diffusion feature of small molecules(inorganic phosphorus,glucose,and CMP)which could through the capsule,and preparation immobilized yeast for more suitable of fermentation,the results showed that the ratio of the wet brewer’s yeast and polyanionic was 0.3,the immobilized yeast and yeast wet mass ratio was 2.5:1 on this condition.Secondly,biosynthesis of CTP by the free yeast.CMP is converted to CTP by Saccharomyces Cerevisiae that has been studied in this paper.By the separation yeast and study about the amount of yeast to raise the synthesis of CTP.Resulting:In this experiment 250 mL-Erlenmeyer flask with 20 mL reaction liquid included 40 mmol/L CMP,150 mmol/L glucose,150 mmol/L phosphate buffer,10 mmol/L magnesium chloride,pH 6.5 was used,and were added 15g beer yeast by 3hours incub ation at 35℃.It need stand 7 h after the separation of yeast.About 85%CMP was transformed to CTP.Finally,CMP is converted to CTP by SA/NaCS-PDMDAAC microcapsules immobilized yeast cells.By the fed-batch glucose to raise the synthesis of CTP.Resulting:In this experiment 250 mL-Erlenmeyer flask with 20 mL reaction liquid included 60 mmol/L CMP,150 mmol/L glucose,250 mmol/L phosphate buffer,20 mmol/L magnesium chloride,pH7 was used,and were added 25g immobilized yeast by 2hours incubation at 35 ℃,then added 0.27g glucose per reaction 2h,when added 2 times,that the total reaction time was 6 h.About 80%CMP was transformed to CTP.The immobilized yeast cells can be repeatedly used 5 times,and the conversion rate remained stable at around 70%. |
