Study On Nucleoside Triphosphates Production Using Beer Yeasts

Using bear yeast enzymes to produce nucleosides triphosphate (NTPs) fromnucleosides monophosphate (NMPs) is one of the most promising techniques in NTPsindustry. Currently, some production problems are eager to be solved , such asoptimizing the producing process, promoting the yields, decreasing costs,masteringreaction mechanism and so on. Beginning with basic research, the reaction mecha-nism has been discussed in this paper. A strain of yeast which has high NTPsproducing activity was selected to study the effect of yeast amount, glucose, inorganicsalts, inorganic phosphates, surfactants, temperature, pH, etc. on the accumulation ofNTPs, and the optimization has been performed..The effect of the multifold factors on NMPs conversion was researched. And theresults showed that the transformation of NMPs to NTPs by yeast enzymes is affectedby kinds of factors. The first process of synthesizing NTP is glycolysis andposphorylation of glucose, and the second process is the accumulation of NTP. Mg2+is the active factor of Embden-Meyerhof Passway (EMP), and excessive addition ofMg2+ will not affect the reaction velocity almost. While the synthesis of CTP, GTPand UTP is based on the formation of ATP. The corresponding NMPs are transformedinto NTPs by the action of nucleoside monophosphate kinase and nucleosidediphosphate kinase, and ATP is served as a phosphoryl donor.The effect of surfactants on the permeability of yeast cell has been investigated.It is found that after the yeast is pretreated with surfactant properly, the permeabilityand conversion ability of yeast cell has been improved, the reaction time has beenshortened, and the yields promoted.The optimal values of the factors which affect the synthesis are obtained throughsingle factorial analysis. On the basis of which, the synthesizing processes of NTPshave been optimized with the help of experimental design, and the optimal parametersare acquired as follows.(1) the synthesis of ATP: AMP:6.5g/L, glucose: 35g/L, beer yeast: 285g/L, iammonium sulfate:5.0g/L, magnesium sulfate:2.0 g/L, potassium dihydrogenphosphate:0.25M, surfactant I : 47.6ml/L, surfactant II : 3.0g/L, pH:6.8,temperature: 38.0℃.(2) the synthesis of CTP: CMP:6.5g/L, glucose: 57.1g/L, beer yeast: 276.5g/L,ammonium sulfate:2.0g/L, magnesium sulfate:2.0 g/L, sodium chloride: 1.0g/L, potassium dihydrogen phosphate:0.22M, surfactant I:17.2ml/L, surfactantII: 4.0g/L, pH:6.5,temperature : 36.0℃.(3) the synthesis of GTP: GMP:7.16g/L, glucose: 55g/L, beer yeast: 270g/L,ammonium sulfate:1.5g/L, magnesium sulfate:2 g/L, potassium dihydrogenphosphate:27.2g/L, surfactant I : 8ml/L, surfactant II : 1.5g/L, pH:6.74,temperature: 37.4℃.(4) the synthesis of UTP: UMP:7.2g/L, glucose: 51.8g/L, beer yeast: 270g/L,ammonium sulfate:2.0g/L, magnesium sulfate:3.0 g/L, potassium dihydrogenphosphate:0.26M, surfactant I :48.1ml/L, surfactant II : 2.0g/L, pH:6.0,temperature: 37.0℃.The yields corresponding to the optimal conditions are obtained as follows: ATP:99.1%, CTP: 97.2%, GTP: 92.7%, UTP: 88.9%, which are higher than the level (ATP:91%, the other three kinds of NTPs: 78～80%) reported.The NTPs synthesizing processes have been optimized in this paper, and thereaction mechanism discussed. An excited progress has been made in the research.With many problems solved, much experience has been commanded, and thecorresponding theory perfected. This paper established a solid practical and theore-tical basis for subsequent research.