DOI QR코드

DOI QR Code

Co-expression of Gamma-Aminobutyrate Aminotransferase and Succinic Semialdehyde Dehydrogenase Genes for the Enzymatic Analysis of Gamma-Aminobutyric Acid in Escherichia Coli

  • So, Jai-Hyun (Korea Promotion Institute for Traditional Medicine Industry) ;
  • Lim, Yu-Mi (Korea Promotion Institute for Traditional Medicine Industry) ;
  • Kim, Sang-Jun (Department of Natural Science, Republic of Korea Naval Academy) ;
  • Kim, Hyun-Ho (Department of Agricultural Chemistry, Kyungpook National University) ;
  • Rhee, In-Koo (Department of Agricultural Chemistry, Kyungpook National University)
  • Received : 2012.11.23
  • Accepted : 2013.02.19
  • Published : 2013.06.30

Abstract

Gamma-aminobutyric acid (GABA) aminotransferase (gabT) and succinic semialdehyde dehydrogenase (gabD) genes from Pseudomonas fluorescens KCCM 12537 were cloned into a single pETDuet-1 vector and co-expressed in Escherichia coli BL21(DE3) simultaneously. The mixture of both enzymes, called GABase, is the key enzyme for the enzymatic analysis of GABA. The molecular mass of the GABA aminotransferase and succinic semialdehyde dehydrogenase were determined to be 52.8 and 46.7 kDa following computations performed with the pI/Mw program, respectively. The GABase activity between pH 6.0 and 9.0 for 24 h at $4^{\circ}C$ remained over 75%, but under pH 6.0 decreased rapidly. The GABase activity between 25 and $35^{\circ}C$ by the treatment at pH 8.6 for 30 min remained over 80%, but over $35^{\circ}C$ decreased rapidly. When the activity against GABA was defined as 100%, the purified GABase activity against 5-aminovaleric acid having a similar structure to GABA showed 47.7% and GABase activity against ${\beta}$-alanine, ${\varepsilon}$-amino-n-caproic acid, $_L$-ornithine, $_L$-lysine, and $_L$-aspartic acid showed between 0.3 to 2.3%. The GABA content was analyzed with this co-expressed GABase, compared with the other GABase which was available commercially. As a result, the content of GABA extracted from brown rice, dark brown rice, and black rice were $26.4{\pm}3.5$, $40.5{\pm}4.7$ and $94.7{\pm}9.3{\mu}g/g$, which were similar data of other GABase in the error ranges.

Keywords

References

  1. Adeghate E and Ponery AS (2002) GABA in the endocrine pancreas: Cellular localization and function in normal and diabetic rats. Tissue Cell 34, 1-6. https://doi.org/10.1054/tice.2002.0217
  2. Baxter CF and Roberts E (1972) The gamma-aminobutyric acid-${\alpha}$-ketoglutaric acid transaminase of beef brain. Methods Neurochem 3, 1135-9.
  3. Griesmann GE, Chan WY, and Owen M (1982) Rennert: Determination of gamma-aminobutyric acid by reversed-phase high-performance liquid chromatography and pre-column labeling for fluorescence detection. J chromatography 230, 121-4. https://doi.org/10.1016/S0378-4347(00)81437-1
  4. Hagiwara H, Seki T, and Ariga T (2004) The effect of pre-germinated brown rice intake on blood glucose and PAI-1 levels in streptozotocin-induced diabetic rats. Biosci Biotechnol Biochem 68, 444-7. https://doi.org/10.1271/bbb.68.444
  5. Jakoby WB (1962) Enzymes of ${\gamma}$-Aminobutyrate Metabolism. Methods Enzymol 5, 771-4.
  6. Jakoby WB and Scott EM (1959) Aldehyde oxidation III. Succinic semialdehyde dehydrogenase. J Biol Chem 234, 937-40.
  7. Lindgren S and Anden NE (1985) Effect of the normal nerve impules flow on the synthesis and utilization of GABA in the rat substantianigra. J Neural Transmission 61, 21-34. https://doi.org/10.1007/BF01253048
  8. Manuel EU and Ramos JL (2001) Expression of a pseudomonas putida aminotransferase involved in lysine catabolism is induced in the rhizosphere. Appl Environ Microbiol 67, 5219-24. https://doi.org/10.1128/AEM.67.11.5219-5224.2001
  9. Oh SH and Choi WG (2000) Production of the quality germinated brown rices containing high ${\gamma}$-aminobutyric acid by chitosan application. Korean J Biotechnol Bioeng 15, 615-20.
  10. Omori M, Yano T, Okamoto J, Tsushida T, Murai T, and Higuchi M (1987) Effect of anaerobically treated tea (gabaron tea) on blood pressure of spontaneously hypertensive rats. Nippon Nogeikagaku Kaishi 61, 1449-51. https://doi.org/10.1271/nogeikagaku1924.61.1449
  11. Phillips AT (1986) Biosynthetic and catabolic features of amino acid metabolism in Pseudomonas. In The bacteria, J. R. Sokatch (ed.), Orlando Academic Press. pp. 385-438.
  12. Sambrook J, Fritsch EF, and Maniatis T (1989) In Molecular Cloning: A Laboratory Manual, (2nd ed). Boston Cold Spring Harbor Laboratory Press, USA.
  13. Seiler N (1981) Polyamine metabolism and function in brain. Neurochem Res 3, 95-110. https://doi.org/10.1016/0197-0186(81)90027-9
  14. Stanton HC (1963) Mode of action of gamma aminobutyric acid on the cardiovascular system. Arch Int Pharmacodyn 143, 195-200.