Preparation And Application Of Cross-Linked Naringinase Aggregates

Cross-linked enzyme aggregates (CLEAs), a new carrier-free immobilized method, with numerous advantages such as low cost, easy preparation, high enzyme activity recovery, good stability, reusability, had crucial research significance in the field of biological enzyme catalysis. Naringinase, composed by a-L-rhamnosidase (EC 3.2.1.40) and P-D-glucosidase (EC 3.2.1.21), could convert the naringin into prunin (4,5,7-trihydroxy flavonone-7-glucoside) and L-rhamnose by a-L-rhamnosidase, and then continue to hydrolyze prunin into naringenin(4,5,7-trihydroxy flavanone) and glucose by β-D-glucosidase. Prunin had a variety of pharmacological activity, such as bactericidal, anti-oxidation, anti-viral, anti-ischemic, anti-tumor, anti-inflammatory, anti-allergic and played an important role in the treatment of cardiovascular diseases and chronic disease. The application of CLEAs to naringinase immobilization could improve its temperature stability, pH stability, and it had important research values for increasing prunin production, reducing costs, improving enzyme catalytic efficiency, and achieving enzymes reuseable. But up to now, there was no related reports about preparing prunin by cross-linked naringinase aggregates.In this paper, CLEAs was used to immobilize naringinase, and the preparation process of cross-linked naringinase aggregates was optimized. According to cross-linked naringinase aggregates enzymatic characteristics, the conversion of naringin to prunin by cross-linked naringinase aggregate in aqueous phase and organic slovent-aqueous biphasic system was optimized. Taking the prunin, prepared by cross-linked naringinase aggregates as the object, using the polyamide resin to separate prunin, and it provided a reference for preparing and purification the prunin. The major contents were presented as follows:(1) Preparation process and enzymatic characteristics of cross-linked naringinase aggregates were investigated, and the research indicated that took the 2.0 folds fermentation broth capacity’s tert-butanol as the precipitant,2.0% glutaraldehyde as the cross-linking agent to prepare cross-linked naringinase aggregates with the condition of 4.0 ℃, cross-linking for 1.5 hours. And the highest enzyme activity recovery was 80.17%. The enzymatic characteristics research indicated that the optimum temperature and pH were 50℃ and 4.0, the Vmax value was 8.35μmol·mL-1·min-1, the Km value was 87.07mmol/L. Compared with the free enzyme, cross-linked naringinase aggregates had good temperature stability and pH stability. Storing for 33 days in 4℃, it’s residual activity was 80.07%, indicating that cross-linked naringinase aggregates had a good storage stability and repeatability.(2) Cross-linked naringinase aggregate was used to catalyze naringin into prunin in aqueous solution, and the catalytic process was optimized by orthogonal design. The optimum conditions were as follows:substrate concentration 13.79mmol/L, pH 8.0, temperature 65℃, enzyme dosage 18.0g/L, reaction time 12.0h. Under the above conditions, the prunin production was about 13.36mmol/L, and the yield of prunin was 96.87%. The cross-linked aggregates was stable, it could successively convert for three cycles, and the yield of prunin could reach 91.29%.(3) The conversion of naringin to prunin by cross-linked naringinase aggregate in organic solvent/aqueous biphasic system was investigated, then orthogonal design was used to optimize the preparation process. The optimum conditions were as follows: ethyl acetate’s volume fraction was 70%, buffer pH 7.0, substrate concentration 20.69mmol/L, temperature 50℃, enzyme dosage 18.0g/L, reaction time 12.0h. Under the above conditions, the production of prunin was about 19.73mmol/L, and the yield of prunin was 92.94%. The cross-linked aggregates could successively convert for three cycles, and the yield of prunin could reach 85.02%.(4) Taking the prunin, produced by cross-linked naringinase aggregate as the research object to explore the purification process of polyamide resin by static and dynamic experiments. Static experimental results showed that adsorption process tended to be balanced after 4 hours, and the equilibrium adsorption amount was18.09mg/g, as well as, desorption process tended to be balanced after 3.0 hours in the 50% isopropyl alcohol solution, and desorption rate was reached 88.92%. The Langmuir equation could be used to effectively fit the process of adsorbing prunin by polyamide resin. Dynamic experimental results showed when the in-flux rate was 0.5ml/min, sample volume reached 240mL, the best adsorption effcets would be obtain 50% isopropyl alcohol was applied to separate the prunin and naringenin after the sample was to be fully adsorbed. Prunin and naringenin could be effectively separated, and the separated prunin’s purity not less than 98%.