The Research On The Preparation Of 5'-Nucleotides By Enzymolysis Of RNA From Beer Yeast

Nucleotides and its derivatives, playing important roles in restoring central nervous system, circulatory system and urinary system, were widely used in food, pharmaceutical, agricultural industry and could be used in synthesizing antivirus and anticancer medicaments. It had been the focus to develop 5'-nucleotides production economically. Comparing with enzymolysis, fermentation and chemical synthesis, the enzymolysis method indicated superiority than the others and had been the focus of research recently. The process of extracting 5'-phosphodiesterase from barley rootlet and the enzymic hydrolysis of RNA into 5'-mononucleotides was studied detailed in the thesis. The separation technology of four kinds of nucleotides by resins and process of 5'-mononucleotides was also involved. The main results were as follows:1 The optimum extraction condition of 5'-phosphodiesterase from barley rootlet was studied in detail. Conditions of 5'-phosphodiesterase extraction: the degree of barley rootlet comminution, temperature, time, pH value, water proportion, inorganic salt and so on were studied detailed and the optimum condition extracting 5'-phosphodiesterase were obtained by single factor and orthogonal analysis. The mesh screen of barely malt was 100; the water adding proportion was 1:8; the pH value was 6.0; the extraction temperature was 40℃; the extraction time was 3 hours. The average enzyme activity of extraction solution from barely malt was 538.1U/mL. The experiment proved proper Zn2+ could improve thermal stability of 5'-phosphodiesterase.2 The preliminary purification and the properties of 5'-phosphodiesterase had been researched. The process of preheating and the enzymolysis of RNA were studied focusing on the effect of temperature, time, amount of Zn2+in preheating, and the effect of concentration of RNA, enzyme amount, temperature, time, the pH value in enzymolysis of RNA. The optimum conditions of preheating were obtained. The preheating temperature was 60℃; the preheating time was 10 min; and the amount of Zn2+ was 0.008mol/L. The optimum condition of enzymic hydrolyzing was also obtained, under which the hydrolysis rate could reached 92.8%. That was: the concentration of RNA was 1%; enzyme amount was 2000U/gRNA; temperature was 75℃; time was 2h, the pH value was 7.0.3 The process of separating nucleotides with column chromatograph was discussed preliminarily. The parameters of nucleotides separation were studied. The velocity was 0.5mL/min; the pH value was 2.0; H/D of column was 21:1, under which the experiment could separate nucleotides well.The process about enzymolysis preparation of 5'-nucleotide was studied in the thesis, which could provide basis to the development and utilization of nucleotide's and its derivates.