• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.3
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• pp.180-185
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• 2005

Cytosine deaminase (cytosine aminohydrolase, EC 3.5.4.1) stoichiometrically catalyzes the hydrolytic deamination of cytosine and 5-fluorocytosine to uracil and 5-fluorouracil, respectively. Amino acid residues located in or near the active sites of the intracellular cytosine deaminase from chromobacterium violaceum YK 391 were identified by chemical modification studies. The enzymic activity was completely inhibited by chemical modifiers, such as 1mM NBS, chloramine-T, $\rho-CMB,\;\rho-HMB$ and iodine, and was strongly inhibited by 1mM PMSF and pyridoxal 5'-phosphate. This chemical deactivation of the enzymic activity was reversed by a high concentration of cytosine. Furthermore, the deactivation of the enzymic activity by $\rho-CMB$ was also reversed by 1mM cysteine-HCI, DTT and 2-mercaptoethanol. These results suggested that cysteine, tryptophan and methionine residues might be located in or near the active sites of the enzyme, while serine and lysine were indirectly involved in the enzymic activity. The intracellular cytosine deaminase from C violaceum YK 391 was assumed to be a thiol enzyme.

• BMB Reports
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• v.28 no.2
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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 Nutrition and Health
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• v.37 no.10
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• pp.881-887
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• 2004

Elevated plasma homocysteine is an independent risk factor for the development of cardiovascular disease. Exercise is generally believed to reduce the plasma homocysteine levels and therefore, being beneficial for cardiovascular disease (CVD). However, there is a possibility that athletes undergoing strenuous training and competition which increase oxidative stress may suffer from increased plasma homocysteine levels. The purpose of this study was to investigate the influence of endurance training on the plasma concentrations of B vitamins and homocysteine in 23 male adolescent field hockey players. Data collection and blood sampling was performed during the training period and non-training period. Following the training period, significant changes in energy and vitamin B6 intakes were observed in these subjects. Plasma vitamin B2, pyridoxal phosphate (PLP) and homocysteine levels were significantly higher during the training period than non-training period, whereas no difference was observed in plasma folate and vitamin B12 levels. Positive correlation was observed between plasma folate and folic acid intakes. When energy, B vitamin intakes were adjusted there was a significant negative correlation between plasma homocysteine levels and plasma riboflavin, folate and vitamin B12 levels. In conclusion, it is suggested that athletes with oxidative stress by strenuous exercise may need B vitamins since riboflavin, folic acid and vitamin Bl2 were shown to be negatively correlated with plasma homocysteine in athletes during the training period.

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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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.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.515.1-515
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• 1986

Thermostable tryptophanase was extracted from a thermophilie bacterium, strain T which was absolutely symbiotic with strain 5. The enzyme was purified 14.7 fold with 5.8% yield by chromatographies using ion exchange, gel filtration, and hydrophobic interaction columns, followed by high performance liquid chromatography on hydroxyapatite column. The purified enzyme has a molecular weight of approximately 210,000 estimated by gel filtration column chromatography, and the molecular weight of subunit was determined by SDS polyacrylamide gel electrophoresis to be 46,000, which indicates that the native enzyme is made of four homologous subunits. The tryptophanase was stable at 65o0 and the optimum temperature for the enzyme activity for 20 min reaction was 70$^{\circ}C$. The purified enzyme activity for 20 min ieaction was 70$^{\circ}C$. The purified enzyme catalyzed the degradation of L-tryptophan into indole, pyruvate and ammonia in the presence of pyridoxal phosphate. 5-Hydroxy-Ltryptophan, 5-methyl-DL-tryptophan, L-cysteine, S-methyl-L-cysteine, 5-methyl-DL-tryptophan, L-cysteine, S-methyl-Lcysteine, and L-serine were also used as substrates to form pyruvate. The amino acid composition of the tryptophanase was determined, and found to contain a high percentage of hydrophobic amino acids, especially in the proline content, which was much higher than that of Escherichia coli tryptophanase. In addition, the 35N-terminal amino acid sequence of the tryptophanase was completely different from that of E. coli tryptophanase.

• Journal of Microbiology and Biotechnology
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• v.17 no.1
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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.

• Journal of Nutrition and Health
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• v.33 no.3
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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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• v.10 no.4
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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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• v.27 no.9
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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.

• Journal of Microbiology and Biotechnology
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• v.17 no.9
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• pp.1579-1584
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• 2007

5-Aminolevulinate (ALA) synthase (E.C. 2.3.1.37), which mediates the pyridoxal phosphate-dependent condensation of glycine and succinyl-CoA, encoded by the Rhodobacter sphaeroides hemA gene, enables Escherichia coli strains to produce ALA at a low level. To study the effect of the enhanced C4 metabolism of E. coli on ALA biosynthesis, NADP-dependent malic enzyme (maeB, E.C. 1.1.1.40) was coexpressed with ALA synthase in E. coli. The concentration of ALA was two times greater in cells coexpressing maeB and hemA than in cells expressing hemA alone under anaerobic conditions with medium containing glucose and glycine. Enhanced ALA synthase activity via coupled expression of hemA and maeB may lead to metabolic engineering of E. coli capable of large-scale ALA production.