Immobilization Of N-Acetyl-D-Neuraminic Acid Aldolase And Characteristic Of Immobilized Enzyme

[Objective] Produce Neu5Ac aldolase in the Fermentation of a inductive strain E.coli DH5α/pRYl and immobilize it, then compare the characteristics of immobilized and free enzyme, laying the ground work for applying immobilized Neu5Ac aldolase in industrial processing. [Methods] The inductive strain E.coli DH5a/pRYl was fermented in the 30L fermentor containing TB broth. Fermenting condition was : Air 1.4vvm, Pug mill 800rpm, 37℃ for beginning l²h, then cool down to 22 ℃.Applied with desired amount of lactose, at 22℃ for 18²0 hours. Then stop fermentation, suspension was centrifuged at 4℃,5000rpm, collect cells. Cells are broken up by Ultrasonic and suspension is centrifuged at 4℃,15000rpm, the supernate is free enzyme. Thermal purification was carried out at 50℃,15min. Free enzyme is immobilized by Eupergit^? C at different ratio of protein/beads, temperature, and time.Then figure out the optimum condition of immobilization. Compare the characteristics of immobilized and free enzyme, including enzymatic kinetics, optimum reactive temperature and pH, thermal and pH stability. Reserving and reusing stability of immobilized Neu5Ac aldolase was studied.[Results] The yield of wet cell was 723g. Immobilized. The optimum condition for Ultrasonic was 700W, 9min. The optimum condition for immobilizing was: protein/beads= lOOmg/g, 20 °C ,24h. Under this condition, immobilized Neu5Ac aldolase with the activity of 10.4U/g was prepared. It was demonstrated that the optimum reaction temperatures was 65 °C for immobilized aldolase and 70°C for free form. Both of them showed the same optimum pH7.5. The value of Km and Vmax for the immobilized Neu5Ac aldolase were 183.7mM/L and 13.67umol/min.mL.However,the free form gave the Km value and Vmax of 59.0mM/L and 7.16|nmol/min.mL. [Conclusion]1. Thermal purification can make Neu5Ac aldolase 2.93 times purified.2. The optimum condition for immobilizing was: protein/beads= lOOmg/g, 20°C,24h. Under this condition, immobilized Neu5Ac aldolase with the activity of 10.4U/g3. The value of Km and Vmax for the immobilized Neu5Ac aldolase were 183.7mM/L and 13.67umol/min.mL.However,the free form gave the Km value and Vmax of 59.0mM/L and 7.16|j.mol/min.mL.4. Compared with free enzyme, immobilized Neu5Ac aldolase shows more stability at a wide range of temperature and pH, which was helpful for industrial application. The optimum temperature and pH of immobilized aldolase was 65 °C and pH7.5 respectively.5. Immobilized aldolase prepared had a good reserving and reusing stability.