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Expression, purification, and characterization of glutamate decarboxylase from human gut-originated Lactococcus garvieae MJF010

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Abstract

Human gut-originated lactic acid bacteria were cultivated, and high γ-aminobutyric acid (GABA)-producing Lactococcus garvieae MJF010 was identified. To date, despite the importance of GABA, no studies have investigated GABA-producing Lactococcus species, except for Lc. lactis. A recombinant glutamate decarboxylase of the strain MJF010 (rLgGad) was successfully expressed in Escherichia coli BL21(DE3) with a size of 53.9 kDa. rLgGad could produce GABA, which was verified using the silylation-derivative fragment ions of GABA. The purified rLgGad showed the highest GABA-producing activity at 35 °C and pH 5. rLgGad showed a melting temperature of 43.84 °C. At 30 °C, more than 80% of the activity was maintained even after 7 h; however, it rapidly decreased at 50 °C. The kinetic parameters, Km, Vmax, and kcat, of rLgGad were 2.94 mM, 0.023 mM/min, and 12.3 min− 1, respectively. The metal reagents of CaCl2, MgCl2, and ZnCl2 significantly had positive effects on rLgGad activity. However, most coenzymes including pyridoxal 5′-phosphate showed no significant effects on enzyme activity. In conclusion, this is the first report of Gad from Lc. garvieae species and provides important enzymatic information related to GABA biosynthesis in the Lactococcus genus.

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References

  • Auteri M, Zizzo MG, Serio R (2015) GABA and GABA receptors in the gastrointestinal tract: from motility to inflammation. Pharmacol Res 93:11–21

    Article  CAS  PubMed  Google Scholar 

  • Cattabeni F, Galli C, Eros T (1976) A simple and highly sensitive mass fragmentographic procedure for γ-aminobutyric acid determinations. Anal Biochem 72:1–7

    Article  CAS  PubMed  Google Scholar 

  • Choi SI, Lee JW, Park SM, Lee MY, Ji GE, Park MS, Heo TR (2006) Improvement of γ-aminobutyric acid (GABA) production using cell entrapment of Lactobacillus brevis GABA 057. J Microbiol Biotechnol 16:562–568

    CAS  Google Scholar 

  • Cotter PD, Hill C (2003) Surviving the acid test: Response of Gram-positive bacteria to low pH. Microbiol Mol Biol Rev 67:429–453

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cui Y, Miao K, Niyaphorn S, Qu X (2020) Production of gamma-aminobutyric acid from lactic acid bacteria: A systematic review. Int J Mol Sci 21:995

    Article  CAS  PubMed Central  Google Scholar 

  • Diana M, Quílez J, Rafecas M (2014) Gamma-aminobutyric acid as a bioactive compound in foods: a review. J Funct Foods 10:407–420

    Article  CAS  Google Scholar 

  • Diez-Gutiérrez L, San Vicente L, Barron LJR, del Carmen Villaran M, Chávarri M (2020) Gamma-aminobutyric acid and probiotics: Multiple health benefits and their future in the global functional food and nutraceuticals market. J Funct Foods 64:103669

    Article  Google Scholar 

  • Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M, Gill SR, Nelson KE, Relman DA (2005) Diversity of the human intestinal microbial flora. Science 308:1635–1638

    Article  PubMed  PubMed Central  Google Scholar 

  • El Kaoutari A, Armougom F, Gordon JI, Raoult D, Henrissat B (2013) The abundance and variety of carbohydrate-active enzymes in the human gut microbiota. Nat Rev Microbiol 11:497–504

    Article  PubMed  Google Scholar 

  • Fan E, Huang J, Hu S, Mei L, Yu K (2012) Cloning, sequencing and expression of a glutamate decarboxylase gene from the GABA-producing strain Lactobacillus brevis CGMCC 1306. Ann Microbiol 62:689–698

    Article  CAS  Google Scholar 

  • Ghatge MS, Contestabile R, di Salvo ML, Desai JV, Gandhi AK, Camara CM et al (2012) Pyridoxal 5′-phosphate is a slow tight binding inhibitor of E. coli pyridoxal kinase. PLoS ONE 7:e41680

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gong L, Ren C, Xu Y (2019) Deciphering the crucial roles of transcriptional regulator GadR on γ-aminobutyric acid production and acid resistance in Lactobacillus brevis. Microb Cell Factories 18:1–12

    Article  CAS  Google Scholar 

  • Hannig G, Makrides SC (1998) Strategies for optimizing heterologous protein expression in Escherichia coli. Trends Biotechnol 16:54–60

    Article  CAS  PubMed  Google Scholar 

  • Heeney DD, Gareau MG, Marco ML (2018) Intestinal Lactobacillus in health and disease, a driver or just along for the ride? Curr Opin Biotechnol 49:140–147

    Article  CAS  PubMed  Google Scholar 

  • Hiraga K, Ueno Y, Oda K (2008) Glutamate decarboxylase from Lactobacillus brevis: activation by ammonium sulfate. Biosci Biotechnol Biochem 72:1299–1306

    Article  CAS  PubMed  Google Scholar 

  • Iwase H, Takeuchi Y, Murai A (1979) Gas chromatography-mass spectrometry of trimethylsilyl derivatives of amino acids. Chem Pharm Bull 27:1307–1315

    Article  CAS  Google Scholar 

  • Kalueff AV, Nutt DJ (2007) Role of GABA in anxiety and depression. Depress Anxiety 24:495–517

    Article  CAS  PubMed  Google Scholar 

  • Kerry RG, Patra JK, Gouda S, Park Y, Shin HS, Das G (2018) Benefaction of probiotics for human health: A review. J Food Drug Anal 26:927–939

    Article  Google Scholar 

  • Kim SH, Shin BH, Kim YH, Nam SW, Jeon SJ (2007) Cloning and expression of a full-length glutamate decarboxylase gene from Lactobacillus brevis BH2. Biotechnol Bioprocess Eng 12:707–712

    Article  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lee B, Lee H, Nam YR, Oh JH, Cho YH, Chang JW (2005) Enhanced expression of glutamate decarboxylase 65 improves symptoms of rat parkinsonian models. Gene Ther 12:1215–1222

    Article  CAS  PubMed  Google Scholar 

  • Lee SJ, Lee HS, Lee DW (2015) Production of γ-aminobutyric acid using immobilized glutamate decarboxylase from Lactobacillus plantarum. Microbiol Biotechnol Lett 43:300–305

    Article  CAS  Google Scholar 

  • Lee KW, Shim JM, Yao Z, Kim JA, Kim HJ, Kim JH (2017) Characterization of a glutamate decarboxylase (GAD) from Enterococcus avium M5 isolated from Jeotgal, a Korean fermented seafood. J Microbiol Biotechnol 27:1216–1222

    Article  CAS  PubMed  Google Scholar 

  • Li H, Qiu T, Huang G, Cao Y (2010) Production of gamma-aminobutyric acid by Lactobacillus brevis NCL912 using fed-batch fermentation. Microb Cell Factories 9:1–7

    Article  Google Scholar 

  • Li H, Li W, Liu X, Cao Y (2013) gadA gene locus in Lactobacillus brevis NCL912 and its expression during fed-batch fermentation. FEMS Microbiol Lett 349:108–116

    Article  CAS  PubMed  Google Scholar 

  • Lim HS, Seo DH, Cha IT, Lee H, Nam YD, Seo MJ (2018) Expression and characterization of glutamate decarboxylase from Lactobacillus brevis HYE1 isolated from kimchi. World J Microbiol Biotechnol 34:1–10

    Article  CAS  Google Scholar 

  • Lin Q, Li D, Qin H (2017) Molecular cloning, expression, and immobilization of glutamate decarboxylase from Lactobacillus fermentum YS2. Electron J Biotechnol 27:8–13

    Article  CAS  Google Scholar 

  • Lyu CJ, Fei JY, Yan JP, Xu QC, Mei JQ, Yue HY et al (2020) Improvement of γ-aminobutyrate biosynthesis by genetically engineered Lactococcus lactis. Biochem Eng J 157:107525

    Article  CAS  Google Scholar 

  • Lyu C, Yao L, Zhu Q, Mei J, Cao Y, Hu S, Zhao W, Huang J, Mei L, Yao S (2021) Reconstruction of the glutamate decarboxylase system in Lactococcus lactis for biosynthesis of food-grade γ-aminobutyric acid. Appl Microbiol Biotechnol 105:4127–4140

    Article  CAS  PubMed  Google Scholar 

  • Madeira F, Lee J, Buso N, Gur T, Madhusoodanan N, Basutkar P, Tivey A, Potter SC, Finn RD, Lopez R (2019) The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res 47:W636–W641

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Murzin AG (1996) Structural classification of proteins: new superfamilies. Curr Opin Struct Biol 6:386–394

    Article  CAS  PubMed  Google Scholar 

  • Niesen FH, Berglund H, Vedadi M (2007) The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc 2:2212–2221

    Article  CAS  PubMed  Google Scholar 

  • Otaru N, Ye K, Mujezinovic D, Berchtold L, Constancias F, Cornejo FA, Krzystek A, de Wouters T, Braegger C, Lacroix C, Pugin B (2021) GABA production by human intestinal Bacteroides spp.: Prevalence, regulation, and role in acid stress tolerance. Front Microbiol 12:656895

    Article  PubMed  PubMed Central  Google Scholar 

  • Park JY, Jeong SJ, Kim JH (2014) Characterization of a glutamate decarboxylase (GAD) gene from Lactobacillus zymae. Biotechnol Lett 36:1791–1799

    Article  CAS  PubMed  Google Scholar 

  • Sa HD, Park JY, Jeong SJ, Lee KW, Kim JH (2015) Characterization of glutamate decarboxylase (GAD) from Lactobacillus sakei A156 isolated from Jeot-gal. J Microbiol Biotechnol 25:696–703

    Article  CAS  PubMed  Google Scholar 

  • Sarasa SB, Mahendran R, Muthusamy G, Thankappan B, Selta DRF, Angayarkanni J (2020) A brief review on the non-protein amino acid, gamma-amino butyric acid (GABA): its production and role in microbes. Curr Microbiol 77:534–544

    Article  CAS  PubMed  Google Scholar 

  • Sender R, Fuchs S, Milo R (2016) Revised estimates for the number of human and bacteria cells in the body. PLoS Biol 14:e1002533

    Article  PubMed  PubMed Central  Google Scholar 

  • Seo MJ, Nam YD, Lee SY, Park SL, Yi SH, Lim SI (2013) Expression and characterization of a glutamate decarboxylase from Lactobacillus brevis 877G producing γ-aminobutyric acid. Biosci Biotechnol Biochem 77:853–856

    Article  CAS  PubMed  Google Scholar 

  • Shin SM, Kim H, Joo Y, Lee SJ, Lee YJ, Lee SJ, Lee DW (2014) Characterization of glutamate decarboxylase from Lactobacillus plantarum and its C-terminal function for the pH dependence of activity. J Agric Food Chem 62:12186–12193

    Article  CAS  PubMed  Google Scholar 

  • Solas M, Puerta E, Ramirez J M (2015) Treatment options in alzheimer s disease: the GABA story. Curr Pharm Des 21:4960–4971

    Article  CAS  PubMed  Google Scholar 

  • Su L, Huang Y, Wu J (2015) Enhanced production of recombinant Escherichia coli glutamate decarboxylase through optimization of induction strategy and addition of pyridoxine. Bioresour Technol 198:63–69

    Article  CAS  PubMed  Google Scholar 

  • Villegas JM, Brown L, de Giori GS, Hebert EM (2016) Optimization of batch culture conditions for GABA production by Lactobacillus brevis CRL 1942, isolated from quinoa sourdough. LWT 67:22–26

    Article  CAS  Google Scholar 

  • Wu Q, Shah NP (2017) High γ-aminobutyric acid production from lactic acid bacteria: emphasis on Lactobacillus brevis as a functional dairy starter. Crit Rev Food Sci Nutr 57:3661–3672

    Article  CAS  PubMed  Google Scholar 

  • Wu Q, Tun HM, Law YS, Khafipour E, Shah NP (2017) Common distribution of gad operon in Lactobacillus brevis and its GadA contributes to efficient GABA synthesis toward cytosolic near-neutral pH. Front Microbiol 8:206

    Article  PubMed  PubMed Central  Google Scholar 

  • Yang SY, Lü FX, Lu ZX, Bie XM, Jiao Y, Sun LJ, Yu B (2008) Production of γ-aminobutyric acid by Streptococcus salivarius subsp. thermophilus Y2 under submerged fermentation. Amino Acids 34:473–478

    Article  CAS  PubMed  Google Scholar 

  • Youn YS, Park JK, Jang HD, Rhee YW (2011) Sequential hydration with anaerobic and heat treatment increases GABA (γ-aminobutyric acid) content in wheat. Food Chem 129:1631–1635

    Article  CAS  Google Scholar 

  • Yu JJ, Oh SH (2011) γ-Aminobutyric Acid Production and Glutamate Decarboxylase Activity of Lactobacillus sakei OPK2-59 Isolated from Kimchi. Korean J Microbiol 47:316–322

    Google Scholar 

  • Yunes RA, Poluektova EU, Dyachkova MS, Klimina KM, Kovtun AS, Averina OV, Orlova VS, Danilenko VN (2016) GABA production and structure of gadB/gadC genes in Lactobacillus and Bifidobacterium strains from human microbiota. Anaerobe 42:197–204

    Article  CAS  PubMed  Google Scholar 

  • Zhang G, Bown AW (1997) The rapid determination of γ-aminobutyric acid. Phytochemistry 44:1007–1009

    Article  CAS  Google Scholar 

  • Huynh K, Partch CL (2015) Analysis of protein stability and ligand interactions by thermal shift assay.Curr Protoc Protein Sci79:28.9.1–28.9.14.

  • Yogeswara IBA, Maneerat S, Haltrich D (2020) Glutamate decarboxylase from lactic acid bacteria-A key enzyme in GABA synthesis. Microorganisms 8:1923

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Acknowledgements

We thank Prof. Jae Kwang Kim (Division of Life Science, Incheon National University) for help in the GC-MS analysis for the GABA production.

Funding

This work was supported by the Incheon National University Research Grant in 2019.

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Author notes

  1. Hyo Jung Lim and Dong-Hyun Jung contributed equally to this work.

Authors and Affiliations

  1. Department of Bioengineering and Nano-Bioengineering, Graduate School of Incheon National University, 22012, Incheon, Republic of Korea

    Hyo Jung Lim, Eui-Sang Cho & Myung-Ji Seo

  2. Microorganism Resources Division, National Institute of Biological Resources, 22689, Incheon, Republic of Korea

    Dong-Hyun Jung

  3. Division of Bioengineering, Incheon National University, 22012, Incheon, Republic of Korea

    Myung-Ji Seo

  4. Research Center for Bio Materials & Process Development, Incheon National University, 22012, Incheon, Republic of Korea

    Myung-Ji Seo

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  1. Hyo Jung Lim
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Contributions

All authors analyzed the experimental data and discussed the results. HJL and E-SC performed the experiments. HJL, D-HJ, and M-JS contributed to the writing of the manuscript.

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Correspondence to Myung-Ji Seo.

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All applicable international, national, and/or institutional guidelines for the care and use of human derivatives were followed. The study of human derivatives was approved by the Institutional Review Board of Incheon National University University (IRB 7007971-202006-001 A).

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Lim, H.J., Jung, DH., Cho, ES. et al. Expression, purification, and characterization of glutamate decarboxylase from human gut-originated Lactococcus garvieae MJF010. World J Microbiol Biotechnol 38, 69 (2022). https://doi.org/10.1007/s11274-022-03256-x

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