Study On Immobilization Of Glutamate Decarboxylase From Rice Bran And Preparation Of γ-aminobutyric Acid

As an endogenous enzyme in rice bran, glutamate decarboxylase (GAD) can catalyze thedecarboxylation of glutamate or its sodium salt to enrich γ-aminobutyric acid (GABA). Andas a health food factor, GABA can be added to food or drugs to lower blood pressure, treatepilepsy, anti-aging, and regulate the secretion of hormones, et al. With fresh rice bran as rawmaterial, preparation and immobilization of GAD, enzymatic properties of immobilizedglutamate decarboxylase (IGAD), preparation and purification of GABA by IGAD werestudied in this paper.First, GAD from rice bran was purified by DEAE-Sephrose FF, HW-55F and Superdex200, and magnetic chitosan microspheres were selected as the immobilized carrier material ofGAD. Magnetic chitosan microspheres were prepared through crosslink emulsificationprocess. The appearance of the microspheres was black-brown and bead-like, and the interiorhad multiphase structures. Magnetic chitosan microspheres had the properties such as: theaverage partical size was6.5μm, the apparent density was0.4912g/mL, and the surface areawas391.9m2/g. They had good magnetic responsiveness, whose settlement balance could bereached within2minutes in magnetic field. The immobilization process of rice bran GAD,use magnetic chitosan microspheres as the carrier, was studied in this paper. Optimaltechnological conditions were obtained through the single factor test and the orthogonal test.Conditions obtained were as follows: pH of phosphate buffer used for magnetic chitosanmicrospheres fully swelling was5.2, glutaraldehyde concentration was1.5%, cross-linked for4h, immobilized for5h. Under this condition, the activity recovery rate of IGAD was39.41%.Secondly, enzymatic properties of IGAD were studied. The optimum temperature wasraised to45°C from40°C, and the optimum pH was lowered to5.2. Compared with the freeenzyme, heat resistance and acid-proof performance were improved after immobilization. Itsstorage stability also had been improved greatly. After30d storage at4°C,68.59%of itsenzyme activity still remained. The Kmand Vmaxof IGAD for Glu were52.2mmol/L and1.6447mg/min, for PLP were2.87μmol/L and1.2794mg/min, while the Kmand Vmaxof freeGAD were37.3mmol/L and2.5661mg/min, for PLP were1.12μmol/Land1.5870mg/min.Then, the preparation process of GABA by IGAD was studied. Optimal technologicalconditions were obtained through the single factor test and the orthogonal test, and conditionsobtained were: NaH2PO4-Na2HPO4(pH5.2) was selected as the buffer, ionic strength was0.04mol/L, substrate concentration was0.05mol/L, and responsed for14h. Under thiscondition, the concentration of GABA was2.205mg/mL.Finally, the purification process of GABA was studied by ion exchange method.717chlorine anion exchange resin was used to remove phosphate from GABA solution. Theadsorption speed of717chlorine anion exchange resin was fast and its adsorption effect wasgood on phosphate at pH7.3. Adsorption equilibrium could be reached rapidly within30min,and the phosphate adsorption rate was79.1%.001×10cation exchange resin was used toadsorb GABA. And adsorption equilibrium could be reached rapidly within40min. Theloading fluid was adjusted to pH3.0, and the loading concentration was adjusted to2mg/mL. After absorbed completely, ammonia water (2.0mol/L) was used to elute it. At this condition,92.18%GABA can be recovered, and the concentration of GABA in the solution was90.32%.