• Journal of Microbiology and Biotechnology
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• 제25권5호
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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.

• BMB Reports
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• 제28권2호
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• pp.124-128
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• 1995

Biodegradative threonine dehydratase purified from Serratia marcescens ATCC 25419 was inactivated by the arginine specific modification reagent, phenylglyoxal (PGO) and the lysine modification reagent, pyridoxal 5'-phosphate (PLP). The inactivation by PGO was protected by L-threonine and L-serine. The second order rate constant for the inactivation of the enzyme by PGO was calculated to be 136 $M^{-1}min^{-1}$. The reaction order with respect to PGO was 0.83. The inactivation of the enzyme by PGO was reversed upon addition of excess hydroxylamine. The inactivation of the enzyme by PLP was protected by L-threonine, L-serine, and a-aminobutyrate. The second order rate constant for the inactivation of the enzyme by PLP was 157 $M^{-1}min^{-1}$ and the order of reaction with respect to PLP was 1.0. The inactivation of the enzyme by PLP was reversed upon addition of excess acetic anhydride. Other chemical modification reagents such as N-ethylmaleimide, 5,5'-dithiobis (2-nitrobenzoate), iodoacetamide, sodium azide, phenylmethyl sulfonylfluoride and diethylpyrocarbonate had no effect on the enzyme activity. These results suggest that essential arginine and lysine residues may be located at or near the active site.

• Journal of Microbiology and Biotechnology
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• 제17권1호
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• pp.116-122
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• 2007

An efficient enzyme system for the synthesis of L-tyrosine was developed using a fed-batch reactor with continuous feeding of phenol, pyruvate, and ammonia. A thermo- and chemostable tyrosine phenol-lyase from Symbiobacterium toebii was employed as the biocatalyst in this work. The enzyme was produced using a constitutive expression system in Escherichia coli BL21, and prepared as a soluble extract by rapid clarification, involving treatment with 40% methanol in the presence of excess ammonium chloride. The stability of the enzyme was maintained for at least 18 h under the synthesis conditions, including 75 mM phenol at pH 8.5 and $40^{\circ}C$. The fed-batch system (working volume, 0.51) containing 1.0 kU of the enzyme preparation was continuously fed with two substrate preparations: one containing 2.2 M phenol and 2.4 M sodium pyruvate, and the other containing 0.4 mM pyridoxal-5-phosphate and 4M ammonium chloride (pH 8.5). The system produced 130g/I of L-tyrosine within 30h, mostly as precipitated particles, upon continuous feeding of the substrates for 22 h. The maximum conversion yield of L-tyrosine was 94% on the basis of the supplied phenol.

• 생명과학회지
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• 제18권4호
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• pp.585-589
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• 2008

Actin cytoskeleton rearrangement를 조절하는 cofilin은 인산기가 제거되면서 활성화되는데 이를 담당하는 효소가 chronophin이다. 이 효소는 비타민 $B_6$의 활성형태인 pyridoxal 5'-phosphate (PLP)의 세포 내 농도를 조절하는 PLP phosphatase로도 알려져 있다. Chronophin은 cap 도메인과 core 도메인을 갖는 HAD family에 속하는 phosphatase이며 다른 HAD phosphatase와 같이 기질결합을 위해 cap 도메인과 core 도메인 사이의 활성부위가 노출되는 열린 형태로의 전환이 있을 것으로 추정되었다. 이전의 밝혀진 chronophin/PLPP의 결정구조에서는 단백질의 결정화과정이 산화된 상태에 이루어졌기에 cap 도메인의 C91과 core 도메인의 C221 사이에 disulfide bond가 있었으며 이것이 cap 도메인과 core 도메인사이의 움직임을 막고 있었다. 본 연구에서는 환원된 상태의 chronophin의 결정체를 얻어 chronophin의 구조를 규명하였다. 환원된 상태의 chronophin의 구조에는 C91과 C221간의 disulfide 결합은 없었으나 산화된 상태와 동일한 닫힌 형태이었으며 국부적인 core 도메인의 움직임이외에는 core 도메인과 cap 도메인의 구조에는 변화가 없었다. 이는 chronophin이 기질이 없는 상태에서 닫힌 형태로 유지되는 것이 disulfide bond에 의한 것이 아님을 의미하며 세포 내의 환원된 상태에서도 닫힌 구조를 유지함으로서 높은 기질 특이성을 보여줄 것임을 암시한다.

• Journal of Microbiology and Biotechnology
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• 제8권6호
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• pp.581-587
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• 1998

Essential amino acids involved in the catalytic role of the extracellular cytosine deaminase from Chromobacterium violaceum YK 391 were determined by chemical modification studies. The enzyme activity required the reduced form of Fe (II) ion, since the enzyme was inhibited by ο-phenanthroline. The enzyme activity was completely inhibited by the chemical modifiers, such as p-chloromercuribenzoate (p-CMB), p-hydroxymercuribenzoate, and chloramine-T at 1 mM each. The enzyme activity was also markedly inhibited by pyridoxal-5'-phosphate, diethyl pyrocarbonate, and phenylmethylsulfonyl fluroride at 1 mM each. The inactivation of the enzyme activity with p-CMB was reversed by a high concentration of cytosine. Furthermore, the inactivation of the enzyme activity with p-CMB was also reactivated by 1 mM dithiothreitol, 1 mM 2-mercaptoethanol, 1 mM cysteine-HCI, 10% ethyl alcohol, and 10% methyl alcohol. These results suggested that cysteine and methionine residues might be located in or near the active site of the enzyme, while lysine, histidine, and serine residues might be indirectly involved in the enzyme activity.

• Journal of Nutrition and Health
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• 제33권3호
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• pp.257-262
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• 2000

This paper is to report our findings that vitamin B6 and folate nutritional state in the rural elderly population with alcohol dependency is poor. The present study was carried out to assess vitamin B6 and folate status in the 17 rural elderly subjects with alcohol dependency and 15 age-and sex-matched controls. Plasma and red cell folate concentrations were analyzed microbiologically, and pyridoxal-5-phosphate dependent erythrocyte alanine aspartate transminase(EAST) activity coefficients were determined using enzyme-coenzyme saturation kinetics. There was no difference in the amount of vitamin consumed between the two groups, and their intakes were 64% and 74.7%, respectively of the Korean dietary recommended allowances for vitamin B6 and folate. The mean percent activation for EAST of the total subjects was greater than 80%, suggesting an inadequate vitamin B6 status between the two groups. Folate concentrations in the red cell, but not in the plasma were significantly lower in the alcohol dependent(141.9ng/ml) subjects than that of the control(233.2ng/ml). Cigarette smokers had lower vitamin B6 and folate levels. Plasma and red cell folate levels were highest among the non-smoking, non-alcohol dependent subjects(11.7 and 257.3ng/ml, respectively) and lowest in the smoker-alcohol dependent group(6.7 and 132.9ng/ml). Finding ways to improve vitamin nutritional state such as vitamin supplementation might be necessary for the rural elderly people, especially for those with alcohol dependency.

• Journal of Nutrition and Health
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• 제10권4호
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• pp.24-32
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• 1977

It was attempted in this study to assess the vitamin $B_{1},\;B_{2}$, and $B_6$ status in tissue by determination of erythrocyte transketolase (TK), glutathione reductase (GR), and aspartate aminotransferase (AST) activities, and their activation by their respective coenzymes, thiamine pyrophosphate, flavin-adenine dinucleotide, and pyridoxal-5-phosphate. The activities of erythrocyte enzymes were stable for more than 30 days when erythrocyte had been stored at $-20^{\circ}C$ and affirmed that the enzyme activities were more stable in the case of deep frozen sotrage of erythrocytes rather than hemolysates. The assay procedures involving ultraviolet kinetic analysis with continuous monitoring for each of enzymes have good within-batch and between-batch precisions and will be avalable in the routine laboratories for the nutritional and clinical surveys. Activity coefficient of TK, GR, and AST was studied in healthy medical students (fifteen men and twelve women, between 21 and 30 years old) on an unrestricted diet. The mean activity coefficient of TK, GR, and AST were 1.18, 1.35, and 2.01 for men, and 1.14, 1.33, and 1.83 for women, respectively. And the upper limit of normal (mean+2SD) were 1.52, 1.69, and 2.61 for men, and 1.50, 1.61, and 2.37 for women, respectively.

• Journal of Nutrition and Health
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• 제27권9호
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• pp.930-939
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• 1994

Vitamin B-6 nutrition has been shown to be inadequate in many population groups including pregnant and lactating women, and in infants. Vitamin B-6 intake was measured in 98 pregnant mothers and a total of 172 cord blood samples of their and other new born infants were analyzed for erythrocyte alanine aminotransferase(EALAT) activities with or without the addition of pyridoxal-5-phosphate to assess vitamin B-6 status of the infants. The average daily vitamin B-6 intake of the pregnant mothers was 1.79mg$\pm$0.88(81.4% of the Recommended Dietary Allowances ; RDA) and vitamin B-6 to protein intake ration was 0.017mg vitamin B-6/g protein. Thirty-eight percent of the pregnant women consumed diets which provided less than the RDA for vitamin B-6 during pregnancy. Seventy-two percent of the dietary pyridoxine intake was provided by the plant food source whose bioavailability was reported to be lower when compared to that of the animal food. The average activity coefficient(AC) values of the cord blood EALAT was 1.41$\pm$0.11, and 32% of the blood samples had EALATAC values greater than 1.25, suggesting that vitamin B-6 status of the newborns might be less than adequate.

• Clinical and Experimental Pediatrics
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• 제58권11호
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• pp.407-414
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• 2015

Early-onset epileptic encephalopathies are one of the most severe early onset epilepsies that can lead to progressive psychomotor impairment. These syndromes result from identifiable primary causes, such as structural, neurodegenerative, metabolic, or genetic defects, and an increasing number of novel genetic causes continue to be uncovered. A typical diagnostic approach includes documentation of anamnesis, determination of seizure semiology, electroencephalography, and neuroimaging. If primary biochemical investigations exclude precipitating conditions, a trial with the administration of a vitaminic compound (pyridoxine, pyridoxal-5-phosphate, or folinic acid) can then be initiated regardless of presumptive seizure causes. Patients with unclear etiologies should be considered for a further workup, which should include an evaluation for inherited metabolic defects and genetic analyses. Targeted next-generation sequencing panels showed a high diagnostic yield in patients with epileptic encephalopathy. Mutations associated with the emergence of epileptic encephalopathies can be identified in a targeted fashion by sequencing the most likely candidate genes. Next-generation sequencing technologies offer hope to a large number of patients with cryptogenic encephalopathies and will eventually lead to new therapeutic strategies and more favorable long-term outcomes.

• Journal of Nutrition and Health
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• 제43권3호
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• pp.315-323
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• 2010

The purpose of this study was to establish the selection of indicators for estimating and factors affecting the requirement of vitamin B6. There has been a need to establish the human requirements of vitamin $B_6$ since vitamin $B_6$ is thought to be involved in more than one hundred biochemical reactions as a coenzyme in the metabolism of amino acids, glucose, and lipid, and the synthesis of neurotransmitters. For the review of the literature, this study included from early findings of the sixties to studies of 2009. This study suggests that plasma pyridoxal 5' phosphate (PLP) is the best single indicator of vitamin $B_6$ status for the healthy but not for the non-healthy. Erythrocyte aspartate aminotransferase and alanine aminotransferase activation by PLP as an indirect measure and urinary 4-pyridoxic acid excretion as a direct measure are useful as supporting indicators. Bioavailability, nutrient interaction, physiological need, and chronic diseases may increase the requirement for vitamin $B_6$. However, these effects can not be quantified due to insufficient evidences.