Purification and Biochemical Properties of Membrane-Bound Lipoxygenase from Sea Algae (Ulva fasciata) and Its Immobilization.

• Main Authors: Wu-Feng Li, 李武峰
• Other Authors: Bonnie Sun Pan
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/55676677277444371293

碩士 === 國立臺灣海洋大學 === 食品科學系 === 94 === Abstract The flavor modification of chicken and fish oil treated with LOX from Ulva（Hu et al., 2000 and Ma et al., 2004）. Because free-form LOX easily affected by environmental factor, LOX activity and stability were lost easily and not be reused. When immobilized the extracts of U. fasciata, the LOX activity were very low. The objective of this study was purified LOX from U. fasciata then to investigate the stability of immobilized LOX, Further the compare and characteristics the biochemical properties from free and immobilized LOX was studied. The recovery of partial purified LOX activities from crude algal LOX extract were 78.9%, 73.7% and purified about 1.77, 3.37-folds by MW10000 membrane and 35-55% saturation of ammonium sulfate fractionation, respectively. The partial purified LOX added 0.2mM CaCl2 during dialysis was retained activity but it was not detected without Ca2+. Adding protease inhibitor PMSF (1mM) and hydroperoxy reducing reagent glutathione (1mM) to partial purified LOX retains 50% of relative activity at 4 days during storage at 4℃.The whole LOX activity was lost within 1 day in the absence of any protector. The lipoxygenase was purified 10-folds from U. fastata using 35-55% saturation of ammonium sulfate fractionation, followed Macroprep Q ion exchange. The recovery of total recovery was 52%. The Michaelis constant ( Km ) of LOX was 117.64、31.25 μM, and maximal velocity ( Vmax ) was 23.25、12.82 μmole hydroperoxy fatty acid/min-mg protein using linoleic acid (C18:2) and arachidonic acid (C20:4) as substrate, respectively. Algal LOX showed the highest activity towards C18:4 followed by C20:4, C18:2 and C18:4-methyled. The partial purified LOX was entrapped using Ca2+-alginate. The immobilized LOX need 110 minutes to reach maximal activity ( A234nm/mg protein ) on shaking rate 100 rpm at 25℃using linoleic acid ( 1μM ) as substrate. When used repeatedly for 6 times, the immobilized LOX was decreased 66% activity. The residual activity of immobilized algal LOX was 51% after a month storage at 4℃. The hydroxy product of arachidonic acid reacted with free and immobilized algal LOX were 15- and 11- hydroxyeicosatetraenoic acid at reactive ratio of 1:7 and 1:6.When linoleic acid was incubated with the LOX, 9-hydroperoxy octadecadienoic acid was found to be main product, and total activity was about 6.63-folds to 13-LOX. Therefore the immobilization of LOX did not affect it’s catalyst specific substrate. To study of the flavor produced by immobilized LOX reacts with different free fatty acid and to identification using GC-MS in the future.