Study On The Properties Of Glutamate Decarboxylase And The Accumulation Of γ-Aminobutyric Acid In Corn Germ

This research was conducted to purify and characterize the GAD from corn germ,to study the enzymology properties of GAD,and to produce corn germ with high GABA content.GAD was purified from corn germ by CM cellulose ion chromatography and SephadexG-75 gel column chromatography.A majority of hybridprotein was separated and the main band from SDS-PAGE showed the subunit Mw was 57k.The purified GAD showed its maximal activity at pH5.7 and 40℃.The enzyme was inactivated completely at 85℃. It was stable at pH4.5-7.5.The Km and Vmax of GAD for Glu was 0.05187mol/L and 1.807mg/min.In addition,GAD could be activated by Ca²⁺,the comparative activity reached 131% when 400μmol/L Ca²⁺ was added,and the apparent Km'was changed to 0.05013mol/L,less than the Km for Glu.This showed that the Ca²⁺ enhanced the affinity between enzyme and Glu,and activated the enzyme.Optimizing the conditions of GABA-accumulation in degreased corn germ by water soaking could increase the content of GABA to 4mg/g, increasing 10 times than before. But a higher GABA content must be obtained using Glu and GAD activator.The optimized conditions were 0.08mol/L phosphate buffer(pH5.7), adding V B6 and CaCl2 3mmol/(molGlu) and 15mmol/(molGlu), the ratio of degreased corn germ to L-Glu was 45g/g, reaction time and temperature were 6h and 40℃. The content of GABA in the final product was 15.572mg/g, the yield was 88.143% and the convert rate of Glu was 100%.