Identification and molecular cloning of glutamate decarboxylase gene from Lactobacillus casei

Document Type: Short communication

Authors

1 Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan, Iran.

2 Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran.

Abstract

Gamma-amino butyric acid (GABA) possesses several physiological functions such as neurotransmission, induction of hypotension, diuretic and tranquilizer effects. Production of GABA-enriched products by lactic acid bacteria has been a focus of different researches in recent years because of their safety and health-promoting specifities. In this study, glutamate decarboxylase (gad) gene of a local strains Lactobacillus casei was identified and cloned. In order to clone the gad gene from this strain, the PCR was carried out using primers designed based on conserved regions. The PCR product was purified and ligated into PGEM-T vector. Comparison of obtained sequences shows that this fragment codes the pyridoxal 5′-phosphate binding region. This strain could possibly be used for the industrial GABA production and also for development of functional fermented foods. Gad gene manipulation can also either decrease or increase the activity of enzyme in bacteria.  

Keywords


1. Hiraga K, Ueno Y, Oda K. Glutamate decarboxylase from Lactobacillus brevis: activation by ammonium sulfate. Biosci Biotech Bioch 2008;72:1299-1306.

2. Bu DF, Erlander MG, Hitz BC, Tillakaratne NJ, Kaufman DL, Wagner-McPherson CB, Evans GA, Tobin AJ. Two human glutamate decarboxylases, 65-kDa GAD and 67-kDa GAD, are each encoded by a single gene. Proc Natl Acad Sci USA 1992;9:2115-2119.

3. Capitani G, Biase DD, Gut CA, Bossa HF, Grütter MG. Crystal structure and functional analysis of Escherichia coli glutamate decarboxylase. EMBO J 2003;22: 4027-4037.

4. Lu X, Xie C, Gu Z. Optimisation of Fermentative Parameters for GABA. Czech J. Food Sci. 2009;27:433-442.

5. Dhakal R, Bajpai VK, Baek KH. Production of gaba (γ-aminobutyric acid) by microorganisms: a review. Brazil J Microbioo 2012;43:1230-1241.

6. Li H, Cao Y. Lactic acid bacterial cell factories for gamma-aminobutyric acid. Amino Acids 2010;39:1107-1116.

7. Siragusa S, Angelis MD, Cagno RD, Rizzello CG, Coda R, Gobbetti M. Synthesis of γ-aminobutyric acid by lactic acid bacteria isolated from a variety of Italian cheeses. Appl Environ Microbiol 2007;73:7283-7290.

8. Kim SH, Shin BH, Kim YH, Nam SW, Jeon SJ. Cloning and expression of a full-length glutamate decarboxylase gene fromLactobacillus brevis BH2. Biotechnol Bioproc Eng 2007;12:707-712.

9. Foerster CW, Foerster HF. Glutamic acid decarboxylase in spores of Bacillus megaterium and its possible involvement in spore germination. J Bacteriol 1973; 114:1090-1098.

10. Smith D, Kassam T, Singh B, Elliott JF. Escherichia coli has two homologous glutamate decarboxylase genes that map to distinct loci. J Bacteriol 1992;174:5820-5826.

11. van Baarlen P, Wells JM, Kleerebezem M. Regulation of intestinal homeostasis and immunity with probiotic lactobacilli. Trends Immunol 2013;34:208-215.

12. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning. Vol. 2. 1989: Cold spring harbor laboratory press New York.

13. Hanahan D. Techniques for transformation of E. coli. DNA clon 1985;1:109-135.

14. Spath K, Heinl S, Grabherr R. Direct cloning in Lactobacillus plantarum: electroporation with non-methylated plasmid DNA enhances transformation efficiency and makes shuttle vectors obsolete. Microb Cell Fact 2012;11:141; 1-8.

15. Lu X, Chen Z, Gu Z, Han Y. Isolation of γ-aminobutyric acid-producing bacteria and optimization of fermentative medium. Biochem Eng J 2008;41:48-52.

16. Huang J, Mei L, Sheng Q, Yao S, Lin D. Purification and characterization of glutamate decarboxylase of Lactobacillus brevis CGMCC 1306 isolated from fresh milk. Chinese J Chem Eng 2007;15:157-161.

17. Komatsuzaki N, Nakamura T, Kimura T, Shima J. Characterization of glutamate decarboxylase from a high γ-aminobutyric acid (GABA)-producer, Lactobacillus paracasei. Biosci Biotech Biochem 2008;72:278-285.

18. Park K-B, Oh S-H. Cloning and expression of a full-length glutamate decarboxylase gene from Lactobacillus plantarum. J Food Sci Nutr 2004;9;324-329.

19. Nomura M, Nakajima I, Fujita Y, Kobayashi M, Kimoto H, Suzuki I, Aso H. Lactococcus lactis contains only one glutamate decarboxylase gene. Microbiology 1999;145;1375-1380.

20. Park KB, Oh SH. Cloning, sequencing and expression of a novel glutamate decarboxylase gene from a newly isolated lactic acid bacterium, Lactobacillus brevis OPK-3. Bioresource Technol 2007;98:312-319.

21. Lin Q, Yang S, Lü F, Lu Z, Bie X, Jiao Y, Zou X. Cloning and expression of glutamate decarboxylase gene from Streptococcus thermophilus Y2. J Gen Appl Microbiol 2009;55:305-310.