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，人们注意到GABA是预防阿尔茨海默氏症、减轻压力和影响饮食效果的物质之一。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的测定GABA的酶法。到目前为止，催化GABA氧化脱氨为琥珀酸半醛，同时产生氨和过氧化氢的酶还没有报道。GABA+H_2O+O_2→琥珀酸半醛+NH_3+H_2O_2在本研究中，我们从土壤中分离到了一种新的GABA氧化酶。经硫酸铵分级和辛基琼脂糖柱层析、DEAE琼脂糖柱层析和羟基磷灰石柱层析，酶的部分纯化倍数为590倍，得率为13.2%。该酶的最适pH为8.0时，酶在pH 7~9范围内稳定15min，最适pH低于40℃。经聚丙烯酰胺凝胶电泳法测定，部分纯化的酶的相对分子质量约为500 kDa。该酶对氨基丁酸表现出较强的活性，而对甘氨酸、β-丙氨酸、5-氨基戊酸和6-氨基己酸等其他化学物质没有活性。测得该酶对GABA的米氏常数(K_M)约为3.14×10~(-2)M，对GABA在1~3 mm范围内呈良好的线性关系。采用GABA氧化酶法测定酸奶中的GABA。该方法适用于食品和生物材料中GABA的测定。使用K_m较低的GABA酶可以提高该方法的灵敏度，因此，我们目前正在研究筛选K_m较低的GABA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.