| Researches were made in this dissertation to improve the preparation and purification of 5'-phosphodiesterase and innovate the production process for the enzymatic hydrolysis of yeast RNA with this enzyme to produce 5'-nucleotides. A new method for the assay of 5'-phosphodiesterase enzyme activity was set up, a method for the isolation and purification of 5'-phosphodiesterase from barley roots was developed, and the dynamic mechanism of the enzymatic hydrolysis reaction was studied, furthermore, a new method for the immobilization of 5'-phosphodiesterase from barley roots was studied, and this immobilized enzyme was applied in a membrane reactor for the hydrolysis of yeast RNA. A continuous cross-flow chromatograph system was invented to separate complexes mixtures, such as products of enzymatic hydrolysis reactions. The main results of this research work were listed as follows:A novel HPLC-RNA method has been developed for the determination of enzyme activity; this method has some advantages compared with the conventional UV-RNA method:a) It can be used for the determination of enzyme activity of 5'-phosphodiesterase sample containing other undesired enzymes. b) It has fine reproducibility, precision, linearity and large measuring range. The correlation coefficients (r2) and R.S.D for the data was 0.9997 and 5.7%, respectively, when the protein concentrations of 5'-phosphodiesterase were within 0.02?1.20 mg/mL.Optimal conditions for the extraction of 5'-phosphodiesterase from barley rootlets were obtained by using orthogonal assay design. The optimal conditions were as:fineness of barley roots was 120 mesh, pH= 7, temperature was 10?, ratio of barley roots to water was 1:16 (w/w), and the extraction time was 5 h. Under the condition, the obtained total enzyme activity of the 5'-phosphodiesterase extracted from 1 g barley rootlets was 4470 U, and the enzyme activity of crude 5'-phosphodiesterase was 280 U/mL.Purer 5'-phosphodiesterase with high specific enzyme activity was obtained through ammonium sulphate precipitation, ultrafiltration, dialysis, and Sephadex G-25?Sephadex G-75 chromatography. The specific enzyme activity of the crude enzyme without ammonium sulphate precipitation was 0.00356 mmol/(mgxmin), while the final enzyme had a specific enzyme activity of 0.02137 mmol/(mgxmin),6 times larger than the original one.The reaction mechanisms of enzymatic hydrolysis of RNA catalyzed by 5'-phosphodiesterase were investigated, and the main enzymatic properties of the enzyme were determined. The results showed that the Michaelis constant (Km) was 8.73 mg/mL(with RNA as substrate), maximum reaction velocity (Vmax) was 0.01427 mg/mL·min, optimum pH was 5.0, and optimum temperature was 70?. This 5'-phosphodiesterase was considerable stable within pH 5?7, and temperature 50?70?. Generally, most metal ions were inhibitors for this enzyme, while magnesian ion was an accelerant for 5'-phosphodiesterase.5'-nucleotides in the products acted as inhibitors, immediate removal of 5'-nucleotides from the hydrolysate was favorable for the enzymatic reaction.5'-phosphodiesterase was immobilized on chitosan activated by glutaric dialdehyde. Under optimum conditions, a maximum enzyme recovery of 53.6%was reached, when the temperature for the immobilization was 30?, pH 5.0, and reaction tine was 6 hours. The immobilized enzyme had an optimum pH 5.5, and optimum temperature 75?. Studies also showed that the immobilized enzyme was purer than free 5'-phosphodiesterase, for the hydrolysate contained more 5'-nucleotides and less other impurities. Michaelis constant (Km) for immobilized enzyme was 15.38 mg/mL(with RNA as substrate)A continuous plug flow reactor(CPFR)coupling with a hollow fiber membrane separator was designed for the hydrolysis of RNA with immobilized 5'-phosphodiesterase. The optimization of the enzymatic reaction showed that the optimum reaction temperature was 75?, optimum pH was 5.5, and the concentration of RNA should be no more than 3%. A continuous experiment of hydrolysis of RNA was conducted in this coupling device, and 88.3% yield of 5'-nucleotides was achieved. Results showed that the immobilized 5'-phosphodiesterase was rather stable during the reaction process, for it kept 74.6% of the original enzyme activity after the operation.A novel continuous cross-flow extraction chromatograph system was invented for the separation of mixtures, Mathematical expressions for the chromatograph both in heavy phase and light phase outlets were derived, which were used to prove theoretically that this system could separate on large scale complex mixtures with high resolution continuously. A mixture of 5'-AMP and benzoic acid was separated well in a 6×6 cross-flow extraction chromatograph system; the result was in line with the theoretical prediction. |
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