Lipoxygenase (EC 1. 13. 11. 12) which catalyzes hydroperoxidation of polyunsaturated fatty acids by direct insertion of molecular oxygen was purified from the water soluble extract of rice bran by conventional methods through ammonium sulfate fractionation, gel filtration using Sephadex G-150, and ion exchange column chromatography on TEAE-cellulose. The enzyme was purified 85 fold with the yield of 26%.
It was found that the enzyme showed its optimum pH and temperature at pH 6.8-pH 7.0 and 30℃, respectively. The enzyme was reasonably stable at room temperature and at neutral pH. Molecular weight determined by gel filtration was 100,000.
The enzyme was highly specific to free fatty acid containing cis, cis-1,4-pentadiene in its structure and the reaction product was identified to posess trans, cisconjugated butadiene system and hydroperoxy group. The enzyme activity was reduced markedly in the presence of chelating agents, commercial antioxidants, and copper ion.
Initial velocity patterns obtained by varying both concentrations of linoleate and oxygen were non-parallel type, which suggested that the enzymic hydroperoxidation proceeded through the formation of intermediate ternary complex between the enzyme and two ligand substrates.
Native enzyme showed EPR signal at g 3.0 and 4.3 due to the existence of ferric ion in the enzyme molecule and the trivalent iron was converted to ferrous state as the enzyme reaction proceeded. Involvement of imidazole group during the catalytic action of the enzyme was identified by photooxidation along with kinetic pH-rate profile. Based on the above results mode of rice bran lipoxygenase action was proposed.
미강으로부터 불포화지방산의 산화를 촉매하는 lipoxygenase 를 분리 정제하였다. Ammonium sulfate 분획과 gel filtration 그리고 이온교환수지의 방법을 사용하여 85 배의 정제된 효소를 얻었으며 이 때 수율은 26% 였다. 효소의 역가는 polarographic oxygen electrode 로 산소의 감소량을 측정하거나 234 nm 에서 흡광도가 증가하는것을 재서 측정하였다. 이 효소의 분자량은 약 100,000 이었으며 pH 6.8 - 7.0, 온도 30℃에서 효소의 역가가 가장 높았다. 실온에서는 안정하나 온도를 올림에 따라서 쉽게 효소역가가 파괴됨을 알 수 있었다. 활성화 에너지는 3.5 Kcal/mole 이었고 상용되는 항산화제에 대하여 상당한저해를 받았다. 또한 cis, cis-1, 4-pentadiene 구조를 가지는 유리지방산에 대하여 높은 기질 특이성을 보였으며 산소와 linoleate 에 대한 친화도는 각각 118 uM, 100 uM 이었다. 효소 반응에 의해서 cis, trans 구조의 conjugated butadiene 을 가지는 hydroperoxide가 생성됨을 확인하였으며 chelating agent 에 많이 저해 되는 것으로 보아 금속 ion의 존재를 추정할 수 있었다. 이 효소는 천연 상태로는 Fe(III) 를 가지나 반응이 진행됨에 따라 Fe(II) 로 변화됨을 EPR spectra 를 통하여 알았으며 active site 에는 imidazole 잔기가 있는 것을 확인하였다. 이러한 실험 결과들로 부터 이 효소 반응의 반응 기전에 대하여 가능한 model 을 제시 하였다.
