Development of a high-sensitive method of γ-aminobutyric acid with a novel γ-aminobutyric acid oxidase

γ-氨基丁酸（GABA）是一种众所周知的神经递质。最近，已经注意到GABA是用于预防阿尔茨海默氏综合征、减轻压力的物质之一，并且是对饮食效果有影响的物质。因此，食品和生物材料中GABA的测定具有重要意义。因此，我们提出了一种高灵敏度的GABA酶法测定方法。迄今为止，催化GABA氧化脱氨生成琥珀酸半醛并同时产生氨和过氧化氢的酶几乎未见报道，其反应式为：GABA+H_2O+O_2→琥珀酸半醛+NH_3+ H_2O_2本研究在从土壤中分离的青霉（Penicillium sp.KAIT-M-117）细胞中发现了一种新的GABA氧化酶。通过硫酸铵分级分离和在辛基-琼脂糖、DEAE-Sepharose和羟基磷灰石上的各种柱层析，酶部分纯化了约590倍，产率为13.2%。酶的最适pH为8.0，在pH 7-9和40℃以下的温度下酶稳定性较好。聚丙烯酰胺凝胶电泳测得部分纯化的酶的分子量约为500 kDa。该酶对GABA表现出强活性，而对其他化学物质如甘氨酸、β-丙氨酸、5-氨基戊酸和6-氨基己酸无活性。该酶对GABA的米氏常数（Km）约为3.14×10 ~（-1）<-2>M。GABA的标准曲线在1 mM ~ 3 mM范围内呈良好的线性关系。将GABA氧化酶法应用于酸奶中GABA的测定。该方法适用于食品和生物材料中GABA的定量分析。用K_m较低的GABA氧化酶可提高该方法的灵敏度。因此，我们目前正在研究筛选具有较低K_m的GABA氧化酶。

γ-aminobutyric acid (GABA) is well known as a neurotransmitter. Recently, it has been noted that GABA is one of substances for a prevention of alzheimer's syndrome, a decrease of stress, and the substance having influence on diet effect. Thus, the determination of GABA in food-stuff and biomaterial is important. Therefore, we proposed the enzymatic method for a high-sensitive method of GABA. To date, the enzyme which catalyzes the oxidative deamination of GABA to succinate semialdehyde with the simultaneous production of ammonia and hydrogen peroxide as indicated by the following equation has not been almost reported.GABA+H_2O+O_2→ succinate semialdehyde+NH_3+H_2O_2In this study, we found a novel GABA oxidase in cells of Penicillium sp.KAIT-M-117 originally isolated from soil. The enzyme was partially purified about 590-fold over the cell free extracts with a yield of 13.2% by ammonium sulfate fractionation and various column chromatographies on Octyl-Sepharose, DEAE-Sepharose, and Hydroxyapatite. The optimum pH for GABA activity was found to be at 8.0, and the enzyme was stable at pH 7-9 for 15 min and below 40℃. The molecular mass of a partially purified enzyme was estimated to be about 500kDa on polyacrylamide gel-electrophoresis. The enzyme showed potent activity toward GABA, wheras it was inactive toward other chemicals such as glycine, β-alanine,5-aminopentanoic acid, and 6-aminohexanoic acid. The Michaelis-Menten constant (K_m) of the enzyme for GABA was caluculated to be about 3.14×10^<-2> M. The calibration curve for GABA was linear from 1 mM to 3 mM. The enzymatic method using GABA oxidase was applied to the determination of GABA in yogurt. This enzymatic method will be applicable to quantification of GABA in food-stuff and biomaterial. The sensitivity of this method can be enhanced by using GABA oxidase having a lower K_m. Therefore, we are currently investigating the screening of GABA oxidase having a lower K_m.