Preparation And Characterization Of Immobilized Glutamate Decarboxylase

GAB A is an essential neurotransmitter inhibitor of mammal which has important medicinal value and has been named as "New resource food" by Ministry of Health. GAD is the key enzyme of synthesis of GABA in the organism, so it can be used for biosynthesis of GABA. In order to improve the efficiency and competitiveness of biosynthesis of GABA, in this work, we immobilized GAD in two different ways, and tried to find the best condition for immobilization of GAD. Finally, we studied the various properties of immobilized GAD including:optimum pH and temperature of immobilized GAD, kinetic constant, operation stability and so on, in order to synthetise GABA.Firstly, the optimum conditions for gel formation and GAD activity were studied, including:the effects of concentration of PVA and SA to diameter, mechanical strength, and GAD activity retention; the effects of PVA/SA ratio to GAD activity retention; the effects of PVA-SA/GAD ratio to GAD activity retention and activity recovery; the effects of concentration of CaCl2 to GAD activity retention. The study shows that the best condition is:concentrations of PVA and SA are 9% and 2.5%; PVA/SA ratio(W/W) is 70/30; PVA-SA/GAD ratio(W/V) is 6/1; concentration of CaCl2 is 1%. Then, zymologic characters and kinetic parameter of PVA-SA immobilized GAD were determined, and the operation stability was evaluated. The results of the investigation are:optimum pH and temperature of immobilized GAD is pH4.8 and 50℃; the pH and thermal stability of GAD have remarkable improvement; Km is 13.6 mmol/L; the immobilized GAD keeps 87% value of initial activity after 7 cycles of reuse. All above prove the promising applications of PVA-SA entrapment GAD.Secondly, zymologic characters and kinetic parameter of Ni-NTA immobilized GAD were determined, and the operation stability was evaluated. In this method, the optimum concentration of GAD is 0.6 mg/mL; optimum pH and temperature of immobilized GAD is pH4.8 and 50℃; GAD has the highest stability in 35℃and pH 5.6; Immoblized GAD with Ni-NTA has the same affinity with free GAD; Km is 34.4 mmol/L; the immobilized GAD keeps 75% value of initial activity after 8 cycles of reuse.