Expression and characterization of glutamate decarboxylase from Lactobacillus brevis HYE1 isolated from kimchi |
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Authors: | Hee Seon Lim Dong-Ho Seo In-Tae Cha Hyunjin Lee Young-Do Nam Myung-Ji Seo |
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Affiliation: | 1.Department of Life Sciences,Graduate School of Incheon National University,Incheon,Republic of Korea;2.Research Group of Gut Microbiome,Korea Food Research Institute,Wanju,Republic of Korea;3.Division of Bioengineering,Incheon National University,Incheon,Republic of Korea;4.Department of Bioengineering and Nano-Bioengineering,Graduate School of Incheon National University,Incheon,Republic of Korea |
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Abstract: | A putative gene (gadlbhye1) encoding glutamate decarboxylase (GAD) was cloned from Lactobacillus brevis HYE1 isolated from kimchi, a traditional Korean fermented vegetable. The amino acid sequences of GADLbHYE1 showed 48% homology with the GadA family and 99% identity with the GadB family from L. brevis. The cloned GADLbHYE1 was functionally expressed in Escherichia coli using inducible expression vectors. The expressed recombinant GADLbHYE1 was successfully purified by Ni–NTA affinity chromatography, and had a molecular mass of 54 kDa with optimal hydrolysis activity at 55 °C and pH 4.0. Its thermal stability was determined to be higher than that of other GADs from L. brevis, based on its melting temperature (75.18 °C). Kinetic parameters including Km and Vmax values for GADLbHYE1 were 4.99 mmol/L and 0.224 mmol/L/min, respectively. In addition, the production of gamma-aminobutyric acid in E. coli BL21 harboring gadlbhye1/pET28a was increased by adding pyridoxine as a cheaper coenzyme. |
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