• 한국식품영양과학회지
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• 제27권5호
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• pp.808-812
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• 1998

Melania snail(Semisulcopira bensoni) is used as ingredient in Korean traditional soup and nutritional foods. Generally, lipoxygenase in several food products may produce off-flavors during their processing and storage. Therefore, the inactivation of lipoxygenase is required to make the better extracts from Melania sanil. Also, the quality on freshness of Melania snail may be evaluated by lipoxygenase activity. The lipoxygenae activity was the highest at 40~60% saturation among several concentrations in salting-ouot saturated solution of ammonium sulfate. The partial purification of lipoxygenase was successfully obtained by Sephacryl S-200 gel chromatography. The first peak among three peaks for protein determination showed the highest activity of lipoxygenase in 13~16 fractions among 100 fractions. The highest peak of lipoxygenase activity by ion exchange chromatography was shown at 0.1M NaCl. In the purification step, the specific activity was 20.8U/mg and activity yield was 19.8%. The optimum pH and temperature were pH6.0~8.0 and 3$0^{\circ}C$, respectively. Molecular weight of the lipoxygenase was estimated about 35kDa by SDS-PAGE.

• 한국식품영양과학회지
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• 제22권6호
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• pp.833-838
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• 1993

토양으로 부터 분리한 lipoxygenase inhibitor 생산균주 Penicillium sp.를 배양하여, 배양액으로 부터 저해물질을 분리, 정제하였다. 배양액을 여과한 후 원심상등액을 농축하고, ethanol 침전에 의해 불순물을 제거하였다. Ethanol은 감압농축기로 제가한 후 Dowex 50W, Sephadex G-25, silica gel column 및 HPLC 등으로 single peak를 나타낼때까지 정제하였다. 정제된 본 저해제는 산성 영역에서 비교적 안정하였으며, 열 안정성이 특히 우수하여 $100^{\circ}C$ 2시간의 가열에 활성이 그대로 유지되었다. 분자량은 약 270으로 측정되었으며, $220^{\circ}C~230^{\circ}C$ 부근에서 용융되지 않고 decomposition 되는 성질을 나타내었다. 저해제는 lipoxygenase와 complex를 형성하여 효소를 효율적으로 저해하였다.

• BMB Reports
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• 제33권2호
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• pp.172-178
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• 2000

The lipoxygenase was purified 35 fold to homogeneity from the Korean red potato by an ammonium sulfate precipitation and DEAE-cellulose column chromatography. The simple purification method is useful for the preparation of pure lipoxygenase. The molecular weight of the enzyme was estimated to be 38,000 by SDS-polyacrylamide gel electrophoreses and Sepharose 6B column chromatography. The purified enzyme with 2 M $(NH_4)_2SO_4$ in a potassium phosphate buffer, pH 7.0, was very stable for 5 months at $-20^{\circ}C$. Because the purified lipoxygenase is very stable, it could be useful for the screening of a lipoxygenase inhibitor. The optimal pH and temperature for lipoxygenase purified from the red potato were found to be pH 9.0. and $30^{\circ}C$, respectively. The Km and Vmax values for linoleic acid of the lipoxygenase purified from the red potato were $48\;{\mu}M$ and $0.03\;{\mu}M$ per minute per milligram of protein, respectively. The enzyme was insensitive to the metal chelating agents tested (2 mM KCN, 1 and 10mM EDTA, and 1 mM $NaN_3$), but was inhibited by several divalent cations, such as $Cu^{++}$, $Co^{++}$ and $Ni^{++}$. The essential amino acids that were involved in the catalytic mechanism of the 5-lipoxygenase from the Korean red potato were determined by chemical modification studies. The catalytic activity of lipoxygenase from the red potato was seriously reduced after treatment with a diethylpyrocarbonate (DEPC) modifying histidine residue and Woodward's reagent (WRK) modifying aspartic/glutamic acid. The inactivation reaction of DEPC (WRK) processed in the form of pseudo-first-order kinetics. The double-logarithmic plot of the observed pseudo-first-order rate constant against the modifier concentration yielded a reaction order 2, indicating that two histidine residues (carboxylic acids) were essential for the lipoxygenase activity from the red potato. The linoleic acid protected the enzyme against inactivation by DEPC(WRK), revealing that histidine and carboxylic amino acids residues were present at the substrate binding site of the enzyme molecules.

• 한국식품과학회지
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• 제19권4호
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• pp.295-299
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• 1987

유안염석과 column chromatography 및 gel여과등으로 비활성이 23.4U/mg이 되는 정제된 mungbean lipoxygenase를 12%의 수율로 얻었다. 정제효소의 작용최적 pH는 8.4이었으며 linoleic acid를 기질로 사용하였을 때 Km값은 0.25mM이었다. 정제효소는 pH $5.0{\sim}7.0$범위와 $50^{\circ}C$이하의 온도에서 비교적 안정하였다. 효소의 활성은 항산화제인 NDGA에 의해 현저히 저해되었고 chelating agents에 의해 저해되었다.

• 한국식품과학회지
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• 제19권5호
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• pp.397-402
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• 1987

황산암모늄분획 침전, 이온교환컬럼 크로마토그래프를 이용하여 감자로부터 2개의 Lipoxygenase isozyme(F-I 및 F-II)을 분리정제하고 각각의 isozyme에 대하여 열불활성화 실험을 행하였다. 분리된 isozyme은 polyacrylamide gel 전기 영동상에서 단일밴드를 보였으며 두 isozyme의 최적 pH는 $5.5{\sim}6.0$으로 비슷하였다. 열불활성화 온동 범위인 $50{\sim}60^{\circ}C$에서 F-I 및 F-II의 $D_{65}$값은 각각 13.3min 및 4.3min이었으며 Z 값은 $11.8^{\circ}C$ 및 $10.3^{\circ}C$ 이었다. 또한 각 isozyme의 열역학적인 상수를 절대반응속도식에 따라 구하였다.