• Biomaterials and Biomechanics in Bioengineering
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• 제3권1호
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• pp.37-46
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• 2016

To maintain activity in a coenzyme/enzyme mixture system, such as ${\beta}$-nicotinamide adenine dinucleotide (NADH)/dehydrogenase, the water-soluble 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers as an additive were synthesized and investigated for their stabilizing function. The inhibitor for the NADH/dehydrogenase reaction was spontaneously formed when the NADH was stored in the dehydrogenase solution. Therefore, we hypothesized that if the additive polymer could interact with an inhibitor without any adverse effect on the dehydrogenase, the activity in the NADH/dehydrogenase mixture could be maintained. We selected lactose dehydrogenase (LDH) as the enzyme, and the NADH was dissolved and incubated at $37^{\circ}C$ in the LDH solution containing the polymers. The phospholipid polymers used in this study were poly(MPC) (PMPC), poly(MPC-co-3-trimethylammonium-2-hydroxypropyl methacrylate chloride) (PMQ) and poly[MPC-co-potassium 3-methacryloyloxypropyl sulfonate ($MSO_3$)] ($PMMSO_3$). The poly($MSO_3$) was used as a reference. For the PMQ and $PMSO_3$ aqueous solutions, the activity of the NADH/LDH mixture system decreased with incubation time as the same level or lower than that in the Tris buffered solution in the absence of the polymers. However, for the poly($MPC-co-MSO_3$) ($PMMSO_3$) aqueous solution, the activity of the NADH/LDH mixed system was six times higher than that in the buffered solution even after a 3-days incubation. The LDH activity was 1.5-1.8 times higher in the presence of the $PMMSO_3$ compared with that in the $PMSO_3$ solution. The mixture of two polymers, poly(MPC) and poly($MSO_3$), did not produce any stabilization. Thus, both the MPC and $MSO_3$ units in the polymer chain had important and cooperative effects for stabilizing the NADH/LDH mixture.

• Journal of Microbiology and Biotechnology
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• 제24권12호
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• pp.1636-1643
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• 2014

3-Hydroxybutyryl-CoA dehydrogenase is an enzyme that catalyzes the second step in the biosynthesis of n-butanol from acetyl-CoA, in which acetoacetyl-CoA is reduced to 3-hydroxybutyryl-CoA. To understand the molecular mechanisms of n-butanol biosynthesis, we determined the crystal structure of 3-hydroxybutyryl-CoA dehydrogenase from Clostridium butyricum (CbHBD). The monomer structure of CbHBD exhibits a two-domain topology, with N- and C-terminal domains, and the dimerization of the enzyme was mostly constituted at the C-terminal domain. The mode of cofactor binding to CbHBD was elucidated by determining the crystal structure of the enzyme in complex with $NAD^+$. We also determined the enzyme's structure in complex with its acetoacetyl-CoA substrate, revealing that the adenosine diphosphate moiety was not highly stabilized compared with the remainder of the acetoacetyl-CoA molecule. Using this structural information, we performed a series of site-directed mutagenesis experiments on the enzyme, such as changing residues located near the substrate-binding site, and finally developed a highly efficient CbHBD K50A/K54A/L232Y triple mutant enzyme that exhibited approximately 5-fold higher enzyme activity than did the wild type. The increased enzyme activity of the mutant was confirmed by enzyme kinetic measurements. The highly efficient mutant enzyme should be useful for increasing the production rate of n-butanol.

• Journal of Nutrition and Health
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• 제32권4호
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• pp.369-375
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• 1999

Hyperthroidism in known to alter the activity of a number of enzymes involved in the catabolism of histidine to CO2. 10-Formyltetrahydrofolate dehydrogenase(EC 1.5, 1.6, 10-formyl-THE dehydrogenase) catalyzes the NADP-dependent conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. In previous studies, 10-formyl-THF dehydrogenase purified from rat and pig liver was coidentified with the cytosolic folate-binding protein. In this study, we investigated the effects of feeding thyroid powder (TP) and thiouracil(TU) on the folate-binding properties of 10-formyl-THE dehydrogenase, the uptake of an injected dose of [3H] folate, and the metabolism of labeled folate to pteroylopoly-${\gamma}$-glutamate in rat liver. The initial hepatic uptake(24hr) of the labeled folate dose was higher in TU-rats and slightly higher in TP-rats in controls. With longer time periods, decreased hepatic uptake of labeled folate was observed in TP-animals compared to euthroid animals, and high levels of hepatic uptake of labeled folate were maintained in TU-animals. This data shows that high levels of thyroid hormone decreased the retention of folate in rat liver. Folate polygutamate chain length was shorter in TU-rats than controls, which suggests that thyroid states do not affect the ability to synthesize pteroylpolyglutamates and that folate polyglutamate might be modulated by altered folate pool size. The ability of 10-formyl-THE dehydrogenase to bind folate in rat liver was similar in both TP-and TU-rats although dehydrogenase activity was changed by thyroid sates.

• Journal of the Korean Wood Science and Technology
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• 제50권3호
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• pp.159-166
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• 2022

CO₂ emissions are the primary reason for global warming; hence, biological and chemical technologies for converting CO₂ into useful compounds are being actively studied. Biological methods using enzymes can convert CO₂ under mild conditions. Formate dehydrogenase (FDH) is a representative CO₂ conversion enzyme. Its function was revealed after isolation from bacteria, yeast, and plants. In this study, we evaluated the CO₂ conversion potential of FDH isolated from wood-rotting fungi. After isolating the FDH gene (TvFDH) from Trametes versicolor, we cloned the full-length FDH from T. versicolor and expressed it in a cell-free expression system. The gene encoding TvFDH was identified as 1,200 bp open reading frame (ORF) and the expected molecular weight of the protein was approximately 42 kDa. Overexpression of the recombinant crude protein including TvFDH was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Enzyme activities and metabolite analyses confirmed the efficiency of TvFDH for CO₂ reduction.

• Fisheries and Aquatic Sciences
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• 제14권2호
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• pp.79-88
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• 2011

The sea tangle, Laminaria japonica has long been used in Korea as a folk remedy to promote health. Gamma-amino butyric acid-enriched (5.56% of dry weight) sea tangle was obtained by fermentation with Lactobacillus brevis BJ-20 (FLJ). A suppressive effect of FLJ on carbon tetrachloride-induced hepatotoxicity has been shown previously. Alcohol administration to Sprague-Dawley rats leads to hepatotoxicity, as demonstrated by heightened levels of hepatic marker enzymes as well as increases in both the number and volume of lipid droplets as fatty liver progresses. However, FLJ attenuated alcohol-induced hepatotoxicity and the accumulation of lipid droplets following ethanol administration. Additionally, FLJ increased the activities and transcript levels of major alcoholmetabolizing enzymes, such as alcohol dehydrogenase and aldehyde dehydrogenase, and reduced blood concentrations of alcohol and acetaldehyde. These data suggest that FLJ protects against alcohol-induced hepatotoxicity and that FLJ could be used as an ingredient in functional foods to ameliorate the effects of excessive alcohol consumption.

• 분석과학
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• 제8권4호
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• pp.869-876
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• 1995

The active sites of the nickel and iron-containing enzyme, carbon monoxide dehydrogenase (CODH) from clostridium thermoaceticum were investigated using Electron Paramagnetic Resonance (EPR) technique. CODH exhibits several spectral features called NiFeC, $g_{ave}=1.82$, $g_{ave}=1.86$. FCII signals which are originated from different clusters in this enzyme. CODH is know to catalyze two different kinds of reactions - acetyl-CoA synthesis and CO oxidation. The acetyl-CoA synthesis activity can be followed by monitoring CO/acetyl-CoA exchange. The addition of 1,10-phenanthroline (phen) to CODH selectively destroyed the CO/acetyl-CoA exchange activity and eliminated the NiFeC signal completely. CO oxidation activity and other EPR signals were unaffected. Such behavior demonstrates that CODH has two distinct active sites and that the NiFe complex is only responsible for the CO/acctyl-CoA exchange activity. Phen caused the removal of only 30% of Ni in the NiFe complex ($0.3Ni/{\alpha}{\beta}$) as shown by the quantitative metal analysis. The phen-treated CODH could be reactivated fully by incubation In $Ni^{2+}$ solution. Radioactive $^{63}Ni^{2+}$ was used to quantitate the amount of the $Ni^{2+}$ incorporated into phen-treated enzyme and showed that the amount was the same as the removed by the phen treatment. i.e. $0.3Ni/{\alpha}{\beta}$. This indicates that only 30% of NiFe complexes are labile and responsible for the CO/acctyl-CoA exchange activity, the other 70% are non-labile and have no exchange activity. This is the first clear evidence that the NiFe complex is heterogencous and labile and non-labile Ni sites arc interacting differently with substrates and chelating agents like phen.

• Asian-Australasian Journal of Animal Sciences
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• 제15권3호
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• pp.421-426
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• 2002

The pyruvate dehydrogenase complex (PDC), a member of $\alpha$-keto acid dehydrogenase complex, catalyzes the oxidative decarboxylation of pyruvate with the formation of $CO_2$, acetyl-CoA, NADH, and $H^+$. This complex contains multiple copies of three catalytic components including pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). Two regulatory components (E1-kinase and phospho-E1 phosphatase) and functionally less-understood protein (protein X, E3BP) are also involved in the formation of the complex. In this study, we have partially cloned the gene for E3BP in human. Nine putative clones were isolated by human genomic library screening with 1.35 kb fragment of E3BP cDNA as a probe. For investigation of cloned genes, Southern blot analysis and the construction of the restriction map were performed. One of the isolated clones, E3BP741, has a 3 kb-SacI fragment, which contains 200 bp region matched with E3BP cDNA sequences. The matched DNA sequence encodes the carboxyl-terminal portion of lipoyl-bearing domain and hinge region of human E3BP. Differences between yeast E3BP and mammalian E3BP coupled with the remarkable similarity between mammalian E2 and mammalian E3BP were confirmed from the comparison of the nucleotide sequence and the deduced amino acid sequence in the cloned E3BP. Cloning of human E3BP gene and analysis of the gene structure will facilitate the understanding of the role(s) of E3BP in mammalian PDC.

• 미생물학회지
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• 제24권2호
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• pp.133-140
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• 1986

일산화탄소를 이용하여 자가영양적으로 성장한 Acinetobacter sp. 1 의 세포추출액은 혐 기성 실험조건하에서 thionin, methylene blue, 2,6-dichlorophenol-indophenol둥올 일산화탄소의 산회를 위한 전자수용체로 사용할 수 있었으나 NAD, NADP, FAD, 또는 FMN등은 천자수용체로 이용하지 못하였다. 이 세균에 존재하는 일산화탄소 산화효소는 유도효소로 밝혀졌고, pH 7.5와 $60^{\circ}C$에서 최대의 활성을 나타내었다. 이 효소의 활성화에너지는 6.1kcal/mol (25.5 kJ/mol)이며 일산화탄소에 대한 Km값은 $154{\mu}M$로 밝혀졌다. 그리고 잘 알려진 몇가지 금속 chelat tIng agent와 2가의 양이온들은 이 효소의 활성에 거의 영향을 미치지 않았는데 $Cu^{2+}$ 이온만은 이 효소의 활성을 완전히 억제시켰다. 또한 이 효소는 포도당과 숙신산에 의해 활성이 저해되었으며, hydrogenase의 활성도 나타내었다. 그리고 Acinetobacter sp. 1의 일산화탄소 산화효소는 Pseudomonas carboxydohydrogena의 일산화탄소 산화효소와 연역학적인 연관성이 없는 것으로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제7권4호
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• pp.215-222
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• 1997

The regulatory mechanism of the flux of acetyl-CoA in Alcaligenes eutrophus in unbalanced growth conditions was investigated using a PHB-negative mutant and transformants reintroduced PHB-biosynthesis enzymes through the transformation of cloned phbCAB genes. The PHB-negative mutant was defected absolutly in PHB synthase but partially in ${\beta}$-ketothiolase and acetoacetyl-CoA reductase, and excreted substantial amount of pyruvate to culture broth at late growth phase. The excretion was due to the inhibitory effect of acetyl-CoA on the activity of pyruvate dehydrogenase. The cloned phbC and phbCAB genes were transformed to the PHB-negative mutant strain to reintroduce PHB biosythesis enzymes. Pyruvate excretion could be decreased substantially but not completely by transformation of PHB synthase alone, while pyruvate excretion was ceased by transformation of all three PHB biosynthesis enzymes. To identify the most critical PHB biosynthesis enzyme influencing on the flux of acetyl-CoA, the effect of the variation of PHB biosynthesis enzymes on pyruvate dehydrogenase was investigated. ${\beta}$-Ketothiolase influenced the activity of pyruvate dehydrogenase more sensitively than PHB synthase. ${\beta}$-Ketothiolase, the first step enzyme of PHB biosynthesis that condense acetyl-CoA to acetoacetyl-CoA, seems to be the major enzyme determining the flux of acetyl-CoA to PHB biosynthesis or TCA cycle, and the rate of PHB biosynthesis in A. eutrophus.

• BMB Reports
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• 제45권12호
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• pp.707-712
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• 2012

Human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) have been known to be inhibited by palmitoyl-CoA with a high affinity. In this study, we have performed the cassette mutagenesis at six different Cys residues (Cys59, Cys93, Cys119, Cys201, Cys274, and Cys323) to identify palmitoyl-CoA binding sites within hGDH2. Four cysteine residues at positions of C59, C93, C201, or C274 may be involved, at least in part, in the inhibition of hGDH2 by palmitoyl-CoA. There was a biphasic relationship, depending on the levels of palmitoyl-CoA, between the binding of palmitoyl-CoA and the loss of enzyme activity during the inactivation process. The inhibition of hGDH2 by palmitoyl-CoA was not affected by the allosteric inhibitor GTP. Multiple mutagenesis studies on the hGDH2 are in progress to identify the amino acid residues fully responsible for the inhibition by palmitoyl-CoA.