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

This research was conducted to purify and characterize the GAD from rice germ, to study the properties of GAD, and to produce rice germ with high GABA content.The methods for GABA assay in rice germ was studied. It was suggested that the colorimetry method could not be used because of the determination error. Paper chromatography was a suitable method, and can be used in combination with HPLC for GABA assay in rice germ.GAD was purified from rice germ by ammonium sulfate fractionation, DEAE- Sephrose FF chromatography, Superdex 200 gel filtration, and Glu- Sephrose CL 4B affinity chromatography. Single band from SDS-PAGE showed the subunit Mw was 40k. Similar molecular weight (78k) was obtained by SE-HPLC. It was suggested that the GAD from rice germ had two homological subunits.The purified GAD showed its maximal activity at pH5.6 and 40℃. The enzyme was inactivated completely at 75 ℃. It was stable at pH4.5-8.0. The Km and Vmax of GAD for Glu was 32.3mmol/L and 1.159mg/min; The Km and Vmax of GAD for PLP was 1.7μmol/L and 1.171mg/min.GAD could be activated by Ca2+, the comparative activity reached 145% when 500μmol/L Ca2+ was added, and the apparent Km' was changed to 25.9mmol/L, less than the Km for Glu. This showed that the Ca2+ enhanced the affinity between enzyme and Glu, and activated the enzyme.The purified GAD could be activated by PLP. The enzyme was inactivated by removal of the bond PLP, but it could be renewed completely after PLP adding. Absorbance spectra of GAD showed one weak peak at 420nm, it was produced by the binding of PLP. Fluorescence emission spectra of GAD was characterized by a Tyr peak, and had no change after PLP removed.The modification of DIC and DEPC resulted in a great loss of GAD activity, suggesting that the His and Arg involve in the enzyme active site. The number of the essential Arg was estimated as one. The Glu could protect the modification of DIC, suggesting that the essential Arg may be in the substrate binding site of the enzyme.Circular Dichroism analysis of GAD showed that the ratios of α-helix and β-sheet in the secondary structures were 13.2% and 38.3%. The secondary structures had a little change after PLP removed. The modification of DIC and DEPC resulted in great changes of the secondary structures. The α-helix might be more important to GAD activity.Optimizing the conditions of GABA - accumulation by water soaking and protein hydrolyzing by trypsin could increase the content of GABA in GABA-RG to 615mg/100g and 2.3g/100g. But a higher GABA content must be obtained using Glu and GAD activator. The optimized conditions were 0.08mol/L phosphate buffer (pH5.6),adding 2 mmol/molGlu of PLP and 20mmol/molGlu of Ca2+ , 42.5U/mmolGlu of rice germ, and the ratio of rice germ to solvent was 1:10, reaction time and temperature were 6h and 40℃. The content of GABA in the final product (GABA-RG3) was 20.6g/100g,the convert rate of Glu was 100%.Finally, the new function of GABA-RG on growth hormone (GH) secretion stimulation and exercise performance enhancing of rats were studied. It showed that the GABA-RG stimulated GH secretion and enhanced leg muscle weight of rats obviously. The swimming time burden with 5% weight and climb time of rats were prolonged. The biological index of rats including blood lactic acid , BUN and liver glycogen were also improved.Judging by relevant specifications of governmental health food standards, we can conclude that GABA-RG has anti-fatigue activities.