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

使用新型γ-氨基丁酸氧化酶开发γ-氨基丁酸的高灵敏度方法

基本信息

  • 批准号:
    15560680
  • 负责人:
  • 金额:
    $ 2.43万
  • 依托单位:
  • 依托单位国家:
    日本
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
  • 财政年份:
    2003
  • 资助国家:
    日本
  • 起止时间:
    2003 至 2005
  • 项目状态:
    已结题

项目摘要

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.
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。

项目成果

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MATSUMOTO Kunio其他文献

MATSUMOTO Kunio的其他文献

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{{ truncateString('MATSUMOTO Kunio', 18)}}的其他基金

Mechanisms for malignant tumor progression based on acquisition of 3-D invasiveness and regulation of epithelial morphogenesis
基于 3D 侵袭性获取和上皮形态发生调节的恶性肿瘤进展机制
  • 批准号:
    24300329
  • 财政年份:
    2012
  • 资助金额:
    $ 2.43万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Research on tissue regeneration regulated by Met/HGF receptor ON-OFF regulation
Met/HGF受体ON-OFF调控的组织再生研究
  • 批准号:
    20390077
  • 财政年份:
    2008
  • 资助金额:
    $ 2.43万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Regulation of tissue regeneration and homeostasis through ON-OFFcontrol of the Met/HGF receptor
通过 Met/HGF 受体的 ON-OFF 控制来调节组织再生和体内平衡
  • 批准号:
    18570127
  • 财政年份:
    2006
  • 资助金额:
    $ 2.43万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
Tissue regeneration and homeostasis through functional Met/HGF receptor modification associated with tissue injury.
通过与组织损伤相关的功能性 Met/HGF 受体修饰来实现组织再生和稳态。
  • 批准号:
    13470037
  • 财政年份:
    2001
  • 资助金额:
    $ 2.43万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Vascular regulation by kringles of HGF and molecular cloning of a novel kringle-containing protein
HGF 的三环对血管的调节以及一种新型三环蛋白的分子克隆
  • 批准号:
    11680631
  • 财政年份:
    1999
  • 资助金额:
    $ 2.43万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
Anti-fibrogenic analysis of HGF in intractable organ failures: Clinical potential of HGF as regenerative therapy
HGF 在顽固性器官衰竭中的抗纤维形成分析:HGF 作为再生疗法的临床潜力
  • 批准号:
    11557010
  • 财政年份:
    1999
  • 资助金额:
    $ 2.43万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Roles of HGF and its family molecules in development.
HGF 及其家族分子在发育中的作用。
  • 批准号:
    07044201
  • 财政年份:
    1995
  • 资助金额:
    $ 2.43万
  • 项目类别:
    Grant-in-Aid for international Scientific Research
INDUCTION OF HGF AFTER LIVER INJURIES AND IDENTIFICATION OF INDUCER FOR HGF
肝损伤后 HGF 的诱导以及 HGF 诱导剂的鉴定
  • 批准号:
    03680173
  • 财政年份:
    1991
  • 资助金额:
    $ 2.43万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (C)
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