Expression and characterization of glutamate decarboxylase from Lactobacillus brevis HYE1 isolated from kimchi

Lim, Hee Seon; Seo, Dong-Ho; Cha, In-Tae; Lee, Hyunjin; Nam, Young-Do; Seo, Myung-Ji

doi:10.1007/s11274-018-2427-6

Cited by 35 publications

(30 citation statements)

References 42 publications

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“…Further increase in the PLP concentration did not increase the production of GABA. This result agrees with those of GADs from E. coli NBRC 3806 and Lactobacillus brevis HYE1 [ 17 , 31 ].…”

Section: Resultssupporting

confidence: 89%

“…BLAST analysis of the amino acid sequences of these GADs indicated that they shared 53% or lower identities with previously reported GADs listed in Table 1 . All these three putative GADs contain the amino acid residues GI(V/S) TY(F)D(T)G (245–250, numbering according to the sequence of ScGAD) conserved for the catalytic site of GADs [ 16 , 17 ] in Additional file 1 : Figure S1. The amino acid residues HV(I)DG(A) ASGG (276–283) are highly conserved in PLP-dependent decarboxylases, such as the GAD from Lactobacillus brevis CGMCC 1306 [ 18 , 19 ].…”

Section: Resultsmentioning

confidence: 99%

“…The kinetic parameters were estimated through in vitro reactions containing different MSG concentrations from 5 mM to 100 mM, 0.01 mM PLP, 200 mM Na 2 HPO 4 -citric acid buffer with corresponding optimum pH value, and 50–100 μL of purified enzyme. The K m and V max values were estimated by using double reciprocal via the Lineweaver-Burk plot [ 17 ]. The k cat and k cat /K m were then calculated.…”

Section: Methodsmentioning

confidence: 99%

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Identification of new glutamate decarboxylases from Streptomyces for efficient production of γ-aminobutyric acid in engineered Escherichia coli

et al. 2019

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BackgroundGamma (γ)-Aminobutyric acid (GABA) as a bioactive compound is used extensively in functional foods, pharmaceuticals and agro-industry. It can be biosynthesized via decarboxylation of monosodium glutamate (MSG) or L-glutamic acid (L-Glu) by glutamate decarboxylase (GAD; EC4.1.1.15). GADs have been identified from a variety of microbial sources, such as Escherichia coli and lactic acid bacteria. However, no GADs from Streptomyces have been characterized. The present study is aimed to identify new GADs from Streptomyces strains and establish an efficient bioproduction platform for GABA in E. coli using these enzymes.ResultsBy sequencing and analyzing the genomes of three Streptomyces strains, three putative GADs were discovered, including StGAD from Streptomyces toxytricini NRRL 15443, SsGAD from Streptomyces sp. MJ654-NF4 and ScGAD from Streptomyces chromofuscus ATCC 49982. The corresponding genes were cloned from these strains and heterologously expressed in E. coli BL21(DE3). The purified GAD proteins showed a similar molecular mass to GadB from E. coli BL21(DE3). The optimal reaction temperature is 37 °C for all three enzymes, while the optimum pH values for StGAD, SsGAD and ScGAD are 5.2, 3.8 and 4.2, respectively. The kinetic parameters including Vmax, Km, kcat and kcat/Km values were investigated and calculated through in vitro reactions. SsGAD and ScGAD showed high biocatalytic efficiency with kcat/Km values of 0.62 and 1.21 mM− 1·s− 1, respectively. In addition, engineered E. coli strains harboring StGAD, SsGAD and ScGAD were used as whole-cell biocatalysts for production of GABA from L-Glu. E. coli/SsGAD showed the highest capability of GABA production. The cells were repeatedly used for 10 times, with an accumulated yield of 2.771 kg/L and an average molar conversion rate of 67% within 20 h.ConclusionsThree new GADs have been functionally characterized from Streptomyces, among which two showed higher catalytic efficiency than previously reported GADs. Engineered E. coli harboring SsGAD provides a promising cost-effective bioconversion system for industrial production of GABA.Electronic supplementary materialThe online version of this article (10.1186/s13036-019-0154-7) contains supplementary material, which is available to authorized users.

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“…Further increase in the PLP concentration did not increase the production of GABA. This result agrees with those of GADs from E. coli NBRC 3806 and Lactobacillus brevis HYE1 [ 17 , 31 ].…”

Section: Resultssupporting

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Identification of new glutamate decarboxylases from Streptomyces for efficient production of γ-aminobutyric acid in engineered Escherichia coli

et al. 2019

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“…Based on the sequence analysis, the three deduced GADs belong to the AAT-I superfamily and shared 96-97% sequence identities. Several conserved motifs were found; GETYTG (235-240) is probably the primary catalytic site or substratebinding site, and the HVDAASGG (266-273) motif is highly conserved in PLP-dependent decarboxylases [15,16]. Further, the motif INVSGHKYGLVYPGLGWIIWR (295-315) is a PLP-binding domain, in which the ε-NH 2 of Lys301 forms Schiff bases with the PLP cofactor through an imine linkage.…”

Section: Gene Cloning and Expression Of Putative Gadsmentioning

confidence: 99%

Characterization of Three GADs from Bacillus spp. for Efficient γ-Aminobutyric Acid Production

Sun¹,

Bai²,

Zhang³

et al. 2021

Preprint

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BackgroundGamma-aminobutyric acid (GABA) is an important bio-product used in pharmaceuticals and functional foods and as a precursor of the biodegradable plastic polyamide 4. Glutamate decarboxylase (GAD) converts l-glutamate (l-Glu) into GABA via decarboxylation. Compared with other methods, develop a bioconversion platform to produce GABA is of considerable interest for industrial use.ResultsThree GAD genes were identified from three Bacillus strains and heterologously expressed in Escherichia coli BL21 (DE3). The optimal reaction temperature and pH values for three enzymes were 40 °C and 5.0, respectively. Of the GADs, GADZ11 had the highest catalytic efficiency towards l-Glu (2.19 mM− 1·s− 1). The engineered E. coli strain that expressed GADZ11 was used as a whole-cell biocatalyst for the production of GABA. After repeated use 8 times, the cells produced GABA with an average molar conversion rate of 97% within 8 h.ConclusionsThree recombinant GADs from Bacillus strains have been conducted functional identification. And engineered E. coli strain heterologous expressing GADZ1, GADZ11, and GADZ20 could accomplish the biosynthesis of l-Glu to GABA in a buffer-free reaction at a high l-Glu concentration. The novel engineered E. coli strain has the potential to be a cost-effective biotransformation platform for the industrial production of GABA.

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“…The GABA concentration of GABA-producing strains was determined by a spectrophotometric GABase assay as described [23] with modifications. Briefly, the reaction mixture (190 µL) contained 140 µL of 100 mM K 4 P 2 O 7 buffer (pH 8.6), 30 µL of 4 mM NADP+, 10 µL of GABase (1 unit/mL, Sigma-Aldrich), and 10 µL of the standard solution (GABA) or culture supernatant (see 2.5.).…”

Section: Quantification Of Gaba Productionmentioning

confidence: 99%

Identification, Classification and Screening for γ-Amino-butyric Acid Production in Lactic Acid Bacteria from Cambodian Fermented Foods

Mayrhofer

Yogeswara

et al. 2019

Biomolecules

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Screening for various types of lactic acid bacteria (LAB) that form the biological agent γ-amino-butyric acid (GABA) is important to produce different kinds of GABA-containing fermented foods. So far, no GABA-producing LAB have been reported from Cambodian fermented foods. Most small-scale fermentations and even some industrial processes in this country still rely on indigenous LAB. The application of GABA-producing autochthonous starters would allow the production of Cambodian fermented foods with an additional nutritional value that meet the population’s dietary habits and that are also more attractive for the international food market. Matrix-assisted laser desorption/ionizing time-of-flight mass spectrometry (MALDI-TOF MS) and partial 16S rDNA sequencing were used to identify 68 LAB isolates from Cambodian fermented foods. These isolates were classified and grouped with (GTG)5 rep-PCR, resulting in 50 strains. Subsequently, all strains were investigated for their ability to produce GABA by thin layer chromatography. GABA-positive strains were further analyzed by the GABase assay. Of the six GABA-positive LAB strains—one Lactobacillus futsaii, two Lactobacillus namurensis, and three Lactobacillus plantarum strains—two Lactobacillus plantarum strains produced high amounts of GABA (20.34 mM, 16.47 mM). These strains should be further investigated for their potential application as GABA-producing starter cultures in the food applications.

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Expression and characterization of glutamate decarboxylase from Lactobacillus brevis HYE1 isolated from kimchi

Cited by 35 publications

References 42 publications

Identification of new glutamate decarboxylases from Streptomyces for efficient production of γ-aminobutyric acid in engineered Escherichia coli

Identification of new glutamate decarboxylases from Streptomyces for efficient production of γ-aminobutyric acid in engineered Escherichia coli

Characterization of Three GADs from Bacillus spp. for Efficient γ-Aminobutyric Acid Production

Identification, Classification and Screening for γ-Amino-butyric Acid Production in Lactic Acid Bacteria from Cambodian Fermented Foods

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