The Preparation And Separation Of Single Nucleotide

As the hydrolysate of ribonucleic acid,5'-Nucleotide have been shown a variety of potential applications, including food industry,medical industry,agriculture and so on,so more and more researchers concentrate their attentions on seeking for a financially feasible method of 5'-Nucleotide production.Technology of separation of four kinds of nucleotides from RNA hydrolysates by Ion-exchange chromatography is studied in paper,as well as the hydrolysis of RNA with the enzymatic method,which sets up their technology routines respectively.The factors on production of 5'-Nucleotide by enzymatic hydrolysis method,such as substrate concentration,enzyme amount, reaction temperature,reaction time,sample pH were studied and the optimal conditions are obtained as follows: substrate concentration 1%, enzyme amount 35u/gRNA, reaction temperature 65℃,sample pH value 5.2 and reaction time 48h. The rate of digestion of RNA reaches 85.60% uder the given conditions.Studies on the process of separation of nucleotides from RNA hydrolysates by positive Ion-exchange Chromatography are carried out,including the influence of sample pH,sample quantity,eluant,eluting velocity on separation.The optimum technological conditions are determined as follows:sample pH value is 2.0,sample quantity is 1.0g/L·resin, eluant is distilled water, eluting velocity is 0.15 times as the volume of column/h.Four kinds of 5'-Nucleotides can be primarily separated by twice separation uder the given conditions, with their purity 5'-GMP 96.9%,5'-AMP 93.65%,5'-CMP 91.64%,5'-UMP 76.40%,and their yield 5'-GMP 95.19%,5'-AMP 92.15%,5'-CMP 91.64%,among which 5'-UMP needs to separated and purified futher.The technological conditions of purification of 5'-UMP are reasearched,showing the effect of sample pH and eluant on purification of 5'-UMP. The optimum technological conditions are given as follows:sample pH value is 1.5, sample quantity is 1.0g/L·resin, eluant is a solution of hydrochloric acid with pH4.0, eluting velocity is 0.15 times as the volume of column/h.The purity of 5'-UMP achives 87.65% under the given conditions.