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

2005 Fiscal Year Final Research Report Summary

• Project/Area Number: 15560680
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biofunction/Bioprocess
• Research Institution: Kanagawa Institute of Technology
• Principal Investigator: MATSUMOTO Kunio Kanagawa Institute of Technology, Department of Applied Chemistry, Professor, 工学部, 教授 (40291752)
• Project Period (FY): 2003 – 2005
• Keywords: GABA / γ-aminobutyric acid oxidase / γ-aminobutyric acid / Penicillium

Research Abstract

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_2
In 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.