Enzymatic Production Of 5'-Nucleotides Using Enzyme Membrane Bioreactor

5'-nucleotides have been widely used in pharmaceutical and food industries. Thestudy of nucleotides has been one of hot spots. And more and more nucleotides areneeded in all kinds of industries. Now four mononucleotides only can be produced byfew factories at the same time. And the purity of 5'-nucleotids is too low, as well asthe production is small, which has led to the shortage of raw material and hasrestrained the development of 5'-nucleotides industry. Enzyme membrane bioreactorhas been developed with the prosperity of biochemical engineering and membraneseparation techniques. As a practical use of the theory integrated bio-reactionseparation process, EMBR has its overwhelming virtues. EMBR has been widely usedin food industry, biochemical engineering, pharmaceutics and environmental process.Thus study of the production of 5'-nucleotides by enzymatic hydrolysis of RNA usingenzyme membrane bioreactor has been invested. Preparation and purification ofnuclease P1, preparation of 5'-nucleotides by free and immobilized enzymemembrane bioreactor and separation of 5'-nucleotides were studied in this paper.Medium optimization for nuclease P 1 production from Penicillium citrinum wasstudied by one-factor-at-a-time method, orthogonal method and response surfacemethodology. Maximum enzyme activity was 354 U/ml under the optimumconditions. Enzyme was purified to homogeneity according to SDS-PAGE by thermaldeactivation, ultrafiltration, (NH₄)₂ SO₄ precipitation, phenyl sepharose chromatography, ion-exchange chromatography and gel filtration. This protocol gave93.4-fold purification with specific activity of 1264 U/mg. The optimum temperatureand pH of nuclease p1 was 69℃and 5.4. It showed good stability below 70℃andpH 4.0-7.0. Its activity was activated by Zn²⁺, K⁺ and strongly inhibited by Cu²⁺, Co²⁺,Al³⁺, SDS. K_m for the enzyme was 24.28 mg/ml with RNA as substrate.The declination curve of membrane permeating flux was studied. Various factorsthat influenced membrane permeating flux such as temperature, pressure wereinvestigated. Operational conditions of enzyme membrane bioreactor were optimized.Preparation of 5'-nucleotides by free enzyme membrane bioreactor was studied byone-factor-at-a-time method and response surface methodology. The optimumenzymatic conditions were as followed: substrate concentration 1.25%, zincconcentration 6.0×10^-4 mol/l, substrate pH 5.3, enzyme concentration 0.080 mg/ml,temperature 65℃, acetic acid buffer solution concentration 0.125 mol/l. Underoptimized conditions, 5'-nucleotides concentration was 12.15 mg/ml and theconversion rate was 97.2%.Chitosan microsphere, DEAE cellulose and paper cellulose were selected as thebetter carriers. The immobilization conditions of these methods were investigated.And the characteristics of immobilized nuclease P1 were studied. The optimum pHvalue was changed after immobilization. The optimum temperature was increased by10℃. Thermal and storage stability were also increased. K_m of paper cellulose,chitosan microsphere, DEAE cellulose immobilized enzyme were 5.72,96.58,27.21mg/ml, respectively. And enzyme was also immobilized on membrane by adsorptionand chemical cross-linking. Enzyme immobilized on membrane by cross-linking wasmore suitable.The use stability of the above immobilized enzyme was studied. The enzymeimmobilized on chitosan microsphere was more suitable for the use of membranebioreactor. Its relatively activity was 96% and the conversion rate was 84% after tentimes usage. Preparation of 5'-nucleotides by enzyme immobilized on chitosanmicrosphere in enzyme membrane bioreactor was studied by one-factor-at-a-timemethod and response surface methodology. The optimum enzymatic conditions were as followed: substrate concentration 1.57%, zinc concentration 6.0×10^-4 mol/l,substrate pH 5.4, enzyme concentration 0.080 mg/ml, temperature 72℃, acetic acidbuffer solution concentration 0.125 mol/l. Under optimized conditions, 5'-nucleotidesconcentration was 14.52 mg/ml and the conversion rate was 92.5%.The separation of enzymatic products 5'-nucleotides was investigated. 5'-AMPand 5'-GMP was separated from cation ion exchange resin IRA120. 5'-UMP and5'-CMP was separated from anion ion exchange resin IRA 400. Process conditionsincluding solution pH, nucleotides loaded amount, the velocity and the eluateconcentration were optimized. The four nucleotides were separated thoroughly andthe average yield was over 80%.