• BMB Reports
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• v.33 no.1
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• pp.82-88
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• 2000

A homologous gene to alanine racemase was cloned from a hyperthermophilic bacterium, Aquifex pyrophilus. The cloned gene encodes a protein of 341 amino acids, which has a significant homology to alanine racemase of Bacillus stearothermophilus, Lactobacillus brevis, and E. coli. When the gene was expressed in Escherichia coli, it produced a 40 kDa protein. The purified protein contains one mole pyridoxal 5-phosphate per one mole of protein, which is essential for catalytic activity of alanine racemase. The purified protein catalyzed racemization of L-alanine to D-alanine, or vice versa, indicating that the cloned gene encoded alanine racemase. It also showed significant racemization activity against L-serine and ${\alpha}-aminobutylic$ acid. The A. pyrophilus alanine racemase showed strong thermostability, and it maintained catalytic activity in the presence of organic solvents.

• BMB Reports
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• v.33 no.1
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• pp.43-48
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• 2000

4-Aminobutyrate aminotransferase is a key enzyme of the 4-aminobutyric acid shunt. It converts the neurotransmitter 4-aminobutyric acid to succinic semialdehyde. In order to study the structural and functional aspects of catalytically active Cys residues of pig brain 4-aminobutyrate aminotransferase, we purified the active form in E. coli by coproduction of thioredoxin. The structural arrangement for functional requirements of a dimeric protein using a bifunctional sultbydryl reagent was then characterized, and the spatial proximity between the essential SH groups and a cofactor (pyridoxal-5'-phosphate) binding site was determined. The bifunctional sultbydryl reagent DMDS reacted with the enzyme at the ratio of one molecule per enzyme dimer. This resulted in an approximately 50% loss of enzymatic activity. The spatial proximity of the distance between the essential SH groups and the cofactor-binding site was determined by the energy transfer measurement technique. The result (approximate 20 ${\AA}$) suggested that cross-linking of two sulfhydryl groups with DMDS is not near a PLP binding site.

• Journal of Nutrition and Health
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• v.26 no.7
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• pp.867-886
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• 1993

Vitamin B-6 status parameters of mothers were assessed in relation to th condition of the infant at birth and during the neonatal period. Parameters were assessed at birth and then weekly in 18 mother-infant pairs during the neonatal period ; mothers were supplemented postnatally with 2 or 27 mg PN-HCI/d. Vitamin B-6 inadequacy in the 2mg supplemented group was suggested by the vitamin status parameters. Mothers whose infants had unsatisfactory Apgar scores at 5min after birth(<7) had lower vitamin B-6 status parameters than mothers whose infants were scored satisfactory. Also, infants who scored unsatisfactory at birth and whose mothers were supplemented with the low level of PN had significantly lower vitamin B-6 status parameters at 7 days of age than infants who scored satisfactory. Infants scored unsatisfactory showed some beneficial effects in both vitamin B-6 status and growth associated with the higher level of maternal postnatal vitamin B-6 supplement. In summary, the mother's prenatal and postnatal vitamin B-6 intake were significantly related to the condition of her infant at birth and during the neonatal period, respectively.

• Microbiology and Biotechnology Letters
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• v.24 no.2
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• pp.222-226
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• 1996

The enzymatic synthesis of 3, 4-dihydroxyphenyl-L-alanine (L-DOPA) was examined for the effects of the reaction additives such as sodium borate, alcohol, and organic solvents. The enzyme used was tyrosine phenol-lyase of Citrobacter freundii KCTC 2006 produced in Escherichia coli. The amounts of tyrosine phenol-lyase and pyridoxal-5-phosphate were optimized to 2.0 units/ml and 0.1 mM, respectively, for the synthetic reaction. Sodium borate, a substance that forms a complex with pyrocatechol, reduced the enzyme deactivation by pyrocatechol although it seriously inhibited the enzyme activity. Among the organic solvents tested, dimethylsulfoxide, dimethylformamide, and alcohol increased the productivity of the L-DOPA synthesis. In a reaction system with 5% methanol, L-DOPA concentration increased up to 210 mM after 24 hours, and 77.1% of which was separated as precipitates. The L-DOPA was purified to 99.96% by solubilizing and recrystallyzing the precipitates.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.73-73
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• 1995

We investigated the effect of derivatized phospholipid, P-pyridoxyl dipalmiuylphosphatidylethanolamine (P-pyr-DPPE), on the catalytic activity of purified porcine brain glutamate decarboxylase(GAD) which catalyzes the synthesis of GABA known as major inhibitory neurotransmitter in CNS. When the P-pyr-DPPE was incorporated into dipalmitdylphosphatidylcholine(DPPC) or phosphatidylserine(PS) vesicles, these vesicles enhanced the catalytic activity of GAD. P-pyr-DPPE also interacted with apoglutamate decarboxylase(apoGAD) and produced the free pyridoxal-5-phosphate(PLP) which is the natural cofactor of GAD. This result indicated that apoGAD catalyzed the cleavage reaction of the P-pyridoxyl moiety of the derivatized phopholipid to generate free PLP, and then free PLP bound to the apoGAD resulting in restroration of the catalytic activity of the enzyme.

• BMB Reports
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• v.38 no.6
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• pp.703-708
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• 2005

We cloned and expressed human pyridoxal-5'-phosphate (PLP) phosphatase, the coenzymatically active form of vitamin $B_6$, in Escherichia coli using pET15b vector. Monoclonal antibodies (mAb) were generated against purified human brain PLP phosphatase in mice, and four antibodies recognizing different epitopes were obtained, one of which inhibited PLP phosphatase. The binding affinities of these four mAbs to PLP phosphatase, as determined using biosensor technology, showed that they had similar binding affinities. Using the anti-PLP phosphatase antibodies as probes, we investigated their cross-reactivities in various mammalian and human tissues and cell lines. The immunoreactive bands obtained on Western blots had molecular masses of ca. 33 kDa. Similarly fractionated extracts of several mammalian cell lines all produced a single band of molecular mass 33 kDa. We believe that these PLP phosphatase mAbs could be used as valuable immunodiagnostic reagents for the detection, identification, and characterization of various neurological diseases related to vitamin $B_6$ abnormalities.

• Korean Journal of Microbiology
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• v.35 no.2
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• pp.133-138
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• 1999

Essential amino acids involving in the active site ofthe cytidn~e deruninase from Bncillus subtilis ED 213 were determined by chemical modification studies. Tllc purified cytidine deruninase tiom Booillus subtilis ED 213 required the reduced form of Fe(lI)ion. since the enzyme was inhibited 43% by 1 mnM o-phenanthroline. Whereas the enzyme activity was activated up to 28% by 1 1 ethylenediaminetetraacetic acid. The cytidine deaninase activily was completely inhibited by 1 mM N-bromosuccinimide, chloramine-T, and p-chloromercuribenzoic acid (p-CMB), respectively. The enzyme activity was inhibited 36% by 1 mM pyridoxal-S-phosphale, and 31% by 1 mM l-ethy~-3-(3-dirneIhj~laminoprop}~~)c~bodiiamide and glycine inethyl ester. The enzyme activity was strongly inhibited 68% by 1 \mu$M \rho$-CMB and this inhibition of the enzyme activity with 1 \mu$M \rho$-CMB was completely reactivated by 5 mM cysleine as a reducing agent. We speculaled that tyrosine, methionine, cysteuie and/or serine residues are located ui or near ihe active site of the cytidine deruniuase from Bncilus subrilis ED 213 and indirectly related to lysine and/or glycine.

• The Korean Journal of Food And Nutrition
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• v.23 no.2
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• pp.171-177
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• 2010

The catabolic threonine dehydratase from Serratia marcescens ATCC 25419 was purified to homogeniety using Sephadex G-200 gel filtration and AMP-Sepharose 4B affinity chromatography. The molecular weight of the native enzyme was 120,000 by native pore gradient PAGE. The enzyme was composed of four identical subunits with subunit molecular weights of 30,000 by SDS-PAGE. The Km values of the enzyme for L-threonine with and without AMP were 7.3 and 92 mM, respectively. There were 2 moles of pyridoxal phosphate and 16 moles of free -SH groups per 1 mole of enzyme. The enzyme was inhibited by $\alpha$-ketobutyrate, pyruvate, glyoxylate, and phosphoenol pyruvate(PEP) in the presence of AMP, yet stimulated by cAMP and ADP. For enzyme properties in comparison with S. marcescens, E. coli, and S. typhimurium enzyme, such as the PLP content, number of free sulfhydryl groups, and existence of ADP binding site, the S. marcescens enzyme was more similar to the S. typhimurium enzyme than the E. coli enzyme. Of the three enteric bacteria, the E. coli and S. typhimurium enzyme was increased the activity by ADP and cAMP, respectively, but only the S. marcescens enzyme was increased the activity by both ADP and cAMP. Therefore, the subtle differences in the properties between enzymes from the three enteric bacteria may represent minor structural differences among these enzymes and warrants further study.

• Applied Biological Chemistry
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• v.22 no.4
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• pp.191-197
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• 1979

${\beta}-Tyrosinase$ was purified and crystallized from cells of Escherichia intermedia A-21 grown in a medium supplemented with 0.2% L-tyrosine. Molecular weight of its subunit, Km value and absorption spectra were determined. Crystallization methods were also studied to eliminate any unnecessary procedures. The results obtained were as follows: 1. The purification procedure included ammonium sulfate fractionation, dialysis against potassium phosphate buffer, pH 6.0 and pH 7.0, and DEAE-Sephadex column chromatography. In the column chromatography, 11 mg of protein was applied per ml of DEAE-Sephadex for efficiency. 2. Steps of protamine sulfate treatment and Sephadex G-150 gel filtration could be eliminated for this enzyme from the known procedures. 3. The purified enzyme was dissolved in 0.01M potassium phosphate buffer containing 2-mercaptoethanol, with a concentration of 20mg/ml. Crystalline enzyme, which appears as hexagonal rods, was obtained by adding solid fine powdered ammonium sulfate to the enzyme solution. 4. Absorption maxima of the enzyme appeared at 340 and 430nm when associated with pyridoxal phosphate. 5. Km value of the enzyme for L-tyrosine was $2.31{\times}10^{-4}M$ and the molecular weight of its subunit was determined by SDS-polyacrylamide electrophoresis to be approximately 50,000.

• Journal of Microbiology and Biotechnology
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• v.25 no.5
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• pp.696-703
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• 2015

A gamma-aminobutyric acid (GABA)-producing microorganism was isolated from jeot-gal (anchovy), a Korean fermented seafood. The isolate, A156, produced GABA profusely when incubated in MRS broth with monosodium glutamate (3% (w/v)) at 37℃ for 48 h. A156 was identified as Lactobacillus sakei by 16S rRNA gene sequencing. The GABA conversion yield was 86% as determined by GABase enzyme assay. The gadB gene encoding glutamate decarboxylase (GAD) was cloned by PCR. gadC encoding a glutamate/GABA antiporter was located immediately upstream of gadB. The operon structure of gadCB was confirmed by RT-PCR. gadB was overexpressed in Escherichia coli BL21(DE3) and recombinant GAD was purified. The purified GAD was 54.4 kDa in size by SDS-PAGE. Maximum GAD activity was observed at pH 5.0 and 55℃ and the activity was dependent on pyridoxal 5'-phosphate. The K_m and V_max of GAD were 0.045 mM and 0.011 mM/min, respectively, when glutamate was used as the substrate.