• 미생물학회지
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• 제48권2호
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• pp.156-162
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• 2012

Salmonella typhimurium LT2 (KCTC 2421)로부터 mannitol dehydrogenase (StMDH)로 추정되는 유전자를 클로닝하고, 대장균에서 대량 발현하였다. 이 유전자는 488개의 아미노산 서열(약 54 kDa)을 암호화하는 1,467 bp의 염기로 구성되며, 이미 보고된 long-chain dehydrogenase/ reductase (LDR) 계열 효소들과 약 36%의 아미노산 서열 상동성을 나타내었다. 재조합 StMDH의 최적 반응온도는 $30^{\circ}C$이며, pH 5.0의 조건에서 최대의 D-fructose 환원활성, 그리고 pH 10.0에서 최대의 D-mannitol 산화활성을 보인다. 반면에 glucose, galactose, xylose, arabinose 등의 기질에 대해서는 활성을 보이지 않았다. 이 효소는 $NAD^+$/NADH 존재 하에서만 산화 환원 활성을 가지며, $NADP^+$/NADPH는 조효소로 이용하지 못하였다. 결론적으로 StMDH는 전형적인 $NAD^+$/NADH 의존형의 mannitol dehydrogenase (EC 1.1.1.67)임을 확인하였다.

• 한국미생물·생명공학회지
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• 제41권2호
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• pp.153-159
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• 2013

Sinorhizobium meliloti 1021 (KCTC 2353) 유전체로부터 mannitol dehydrogenase (SmMDH)로 추정되는 유전자를 클로닝하고, 대장균에서 대량 발현하였다. 이 유전자는 494개의 아미노산(약 54 kDa)을 암호화하는 1,485 bp의 염기로 구성되며, 기존에 보고된 long-chain dehydrogenase/reductase 계열 MDH 효소들과 약 35-55%의 아미노산 서열상동성을 나타내었다. 재조합 SmMDH의 최적 반응온도는 $40^{\circ}C$이며, pH 7.0의 조건에서 최대의 D-fructose 환원활성, 그리고 pH 9.0에서 최대의 D-mannitol 산화활성을 보였다. 특히, 이 효소는 $NAD^+/NADH$ 조효소의 존재 하에서 산화 환원 활성을 나타내며, $NAD^+/NADPH$는 조효소로 이용하지 못하였다. 결론적으로 SmMDH는 전형적인 $NAD^+/NADH$-의존형 mannitol dehydrogenase (EC 1.1.1.67)임을 확인하였다.

• KSBB Journal
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• 제11권4호
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• pp.489-496
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• 1996

Carbon paste electrode를 채용함으로써 L-lac tate 측정용의 전기화학식 바이오센서를 성공적으로 개발할 수 있었다. 특히 뛰어난 electrocatalytic activity를 갖는 platinum이 첨가된 platinum-CPE 를 이용하여 낮은 전위에서의 NADH의 전기척 산 화가 가능하였다. Enzyme (lactate dehydrogen­a ase)과 $NAD^+$를 carbon paste형식으로 직접 제조 함으로써 L-lactate 측정을 위한 성공적인 바이요센 서의 개발이 가능하였다. 위와 같은 개발연구를 통 하여 다른 $NAD^+$ -dependent dehydrogenase를 채 용한 바이오센서로의 적용이 기대된다.

• Journal of Microbiology and Biotechnology
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• 제13권4호
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• pp.501-508
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• 2003

Artificial coupling of one enzyme with another can provide an efficient means for the production of industrially important chemicals. Xylose reductase has been recently discovered to be useful in the reductive production of xylitol. However, a limitation of its in vitro or in vivo use is the regeneration of the cofactor NAD(P)H in the enzyme activity. In the present study, an efficient process for the production of xylitol from D-xylose was established by coupling two enzymes. A NADH-dependent xylose reductase (XR) from Pichia stipitis catalyzed the reduction of xylose with a stoichiometric consumption of NADH, and the resulting cofactor $NAD^+$ was continuously re-reduced by formate dehydrogenase (FDH) for regeneration. Using simple kinetic analyses as tools for process optimization, suitable conditions for the performance and yield of the coupled reaction were established. The optimal reaction temperature and pH were determined to be about $30^{\circ}C$ and 7.0, respectively. Formate, as a substrate of FDH, affected the yield and cofactor regeneration, and was, therefore, adjusted to a concentration of 20 mM. When the total activity of FDH was about 1.8-fold higher than that of XR, the performance was better than that by any other activity ratios. As expected, there were no distinct differences in the conversion yields of reactions, when supplied with the oxidized form $NAD^+$ instead of the reduced form NADH, as a starting cofactor for regeneration. Under these conditions, a complete conversion (>99%) could be readily obtained from a small-scale batch reaction.

• Journal of Microbiology and Biotechnology
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• 제14권5호
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• pp.919-923
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• 2004

The sk10 isolated from kimchi was identified as W. kimchii on the basis of l6s-rDNA sequencing. Studies were made to analyze the metabolic flux shift of the sk10 on glucose under aerobic growth conditions. The sk10 produced 38.2 mM acetate, 16.3 mM ethanol, and 33.2 mM lactate under aerobic conditions, but 2.4 mM acetate, 48.0 mM ethanol, and 44.1 mM lactate under anaerobic conditions. The NADH peroxidase (NADH-dependent hydrogen peroxidase) activity of sk10 grown under aerobic conditions was 11 times higher than that under anaerobic conditions. Under the low ratio of $NADH/NAD^+$, the metabolic flux toward lactate and ethanol was shifted to the flux through acetate kinase without NADH oxidation. The kinds of enzymes and metabolites of sk10 were close to those in the pathway of Leuconostoc sp., but the metabolites produced under aerobic growth conditions were different from those of Leuconostoc sp. The stoichiometric balance calculated using the concentrations of metabolites and substrate was about 97%, coincident with the theoretical values under both aerobic and anaerobic conditions. From these results, it was concluded that the metabolic flux of W. kimchii sk10 was partially shifted from lactate and ethanol to acetate under aerobic conditions only.

• Journal of Microbiology and Biotechnology
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• 제23권12호
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• pp.1699-1707
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• 2013

During the fermentative production of 1,3-propanediol under high substrate concentrations, accumulation of intracellular 3-hydroxypropionaldehyde will cause premature cessation of cell growth and glycerol consumption. Discovery of oxidoreductases that can convert 3-hydroxypropionaldehyde to 1,3-propanediol using NADPH as cofactor could serve as a solution to this problem. In this paper, the yqhD gene from Klebsiella pneumoniae DSM2026, which was found encoding an aldehyde reductase (KpAR), was cloned and characterized. KpAR showed broad substrate specificity under physiological direction, whereas no catalytic activity was detected in the oxidation direction, and both NADPH and NADH can be utilized as cofactors. The cofactor binding mechanism was then investigated employing homology modeling and molecular dynamics simulations. Hydrogen-bond analysis showed that the hydrogen-bond interactions between KpAR and NADPH are much stronger than that for NADH. Free-energy decomposition dedicated that residues Gly37 to Val41 contribute most to the cofactor preference through polar interactions. In conclusion, this work provides a novel aldehyde reductase that has potential applications in the development of novel genetically engineered strains in the 1,3-propanediol industry, and gives a better understanding of the mechanisms involved in cofactor binding.

• Journal of Korean Biological Nursing Science
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• 제14권2호
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• pp.129-138
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• 2012

Purpose: Cachexia is a complex metabolic syndrome associated with wasting of skeletal muscle which contributes to nearly one-third of all cancer deaths. Cachexia lowers the frequency of response to chemotherapy and radiation and ultimately can impact survival as well as quality of life during treatment. NF-kappa B is one of the most important molecular mediators of cachexia. In this study, therefore, possible candidates for inhibitors of NF-kappa B were searched. Methods: Amino acids that regulate cellular redox potential by adjusting the level of NAD/NADH ratio, such as aspartate, pyruvate, and isocitrate were selected. Results: Pyruvate effectively inhibited luciferase activity in TNF-stimulated 293T cells transfect with an NF-kB dependent luciferase reporter vector. Pyruvate also showed protective effect on muscle atrophy of differentiated C2C12 myocyte induced by TNF/IFN. Conclusion: We might be able to develop the nutritional management strategy for cancer cachexia patients with pyruvate supplementation.

• BMB Reports
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• 제33권4호
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• pp.321-325
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• 2000

Quinone reductase was purified to homogeneity from bovine liver by using ammonium sulfate fractionation, ionexchange chromatography, and gel filtration chromatography. The enzyme utilized either NADH or NADPH as the electron donor. The enzyme catalyzed the reduction of several quinones and other artificial electron acceptors. Furthermore, the enzyme catalyzed NAD(P)H-dependent reduction of azobenzene. The apparent Km for 1,4-benzoquinone and azobenzene was 1.64 mM and 0.524 mM, respectively. The reduction of azobenzene by quinone reductase was almost entirely inhibited by dicumarol or Cibacron blue 3GA, potent inhibitors of the mammalian quinone reductase. In the presence of 1.0${\mu}M$ Cibacron blue 3GA, azoreductase activity was lowered by 45%, and almost complete inhibition was seen above 2.0 ${\mu}M$ Cibacron blue 3GA.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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• pp.638-639
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• 2000

Quinone reductase was purified to electrophoretic homogeneity from bovine liver by using ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration chromatography. The enzyme utilized either NADH or NADPH as the electron donor. The optimum pH of the enzyme was pH 8.5, and the activity of the enzyme was greatly inhibited by $Cu^{2+}$ and $Hg^{2+}$ ions, dicumarol and cibacron blue 3GA. The enzyme catalyzed the reduction of several quinones and other artificial electron acceptors. Furthermore, the enzyme catalyzed NAD(P)H-dependent reduction of azobenzene or 4-nitroso-N,N-dimethylaniline. The apparent $K_m$ for 1,4-benzoquinone, azobenzene, and 4-nitroso-N,N-dimethylaniline was 1.64mM, 0.524mM and 0.225mM, respectively. The reduction of azobenzene or 4-nitroso-N,N-dimethylaniline by quinone reductase was strongly inhibited by dicumarol or cibacron blue 3GA, potent inhibitors of quinone reductase.

• 생명과학회지
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• 제25권11호
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• pp.1290-1297
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• 2015

에탄올 생산 세균 Zymomonas mobilis에서 에탄올 생산 경로의 핵심으로 작용하는 효소인, pyruvate decarboxylase(pdc) 유전자의 불활성 실험을 통해, PDC 활성이 50% 감소된 PDC 활성 변형균주가 분리되었다. 이러한 균주들의 에탄올 탄소대사 흐름이 고부가가치 화합물인 피루브산, 숙신산 및 젖산 등으로 전환되는지를 발효 실험을 통해 평가하였다. 하지만 pdc의 발현을 중지시키기 위해 cat-삽입형-pdc와 pdc-결손형 아형 유전자를 전기천공법을 이용해 야생형 균주 ZM4의 염색체에 이식하기 위한 다수의 시도에도 불구하고, 이러한 방법을 통해 분리된 균주들은 대부분 부분적 유전자 불활성 특성을 보였으며, PDC 활성이 완전히 손실된 삭제 돌연변이 균주를 획득할 수는 없었다. PDC활성이 변형된 돌연변이 균주의 발효 실험에서, 야생형 균주와 비교 시 감소된 PDC 효소 활성의 변화로 인해 기질 흡수율과 에탄올 생산율이 감소되어 피루브산 생산이 약 2.5 g l^-1 정도로 증가함을 확인하였으나, 젖산과 숙신산의 생산에 현저한 농도 변화를 보이지 못했다. 이러한 결과는 Z. mobilis의 산화환원 에너지가 PDC 효소 활성에 의한 에탄올 생산 경로에 전적으로 의존하여 발생한다는 것을 암시하였다. 상기 결과를 토대로 pdc 유전자의 완전한 불활성 유도와 산화환원 에너지의 균형은, 젖산 생산을 위한 lactate dehydrogenase, 숙신산 생산을 위한 pyruvate dehydrogenase와 malic enzyme과 같은 효소의 활성 증가를 통해, 세포내 NAD와 NADH 농도의 산화환원 균형이 이루어져야 발생할 수 있음을 시사하였다.