The Study Of Purification Of Soybean Lipoxygenase And Its Catalysis Of Polyunsaturated Fatty Acids

Lipoxygenase (Lipoxygenase, EC1.13.11.12, LOX) is an oxidoreductase, as the activity of soybean lipoxygenase is higher than that of other plants, and LOX which is extracted from soybean lipoxygenase is more efficient. Therefore, most studies were based on soybean lipoxygenase. The basic path way of the production of flavor compounds is that fatty acid hydroperoxides was produced in presence of soybean lipoxygenase and O2,then it was cleaved into C6, C9 or the C10 aldehyde by hydroperoxide lyase, as LOX and HPL enzymes with different specificity.In this paper, the extraction and purification of LOX, high purity LOX and the physical, chemical and enzymatic properties of LOX were studied. In the LOX reaction, the aspect of the enzyme reactor and optimization reaction conditions were studied. As the reaction system for the water-oil-gas coexistence and mass transfer mechanism is complicated, the optimal reaction conditions were investigated to improve the rate of mass transfer and seek the best process condition, so that the conversion rate of lipid hydroperoxide was most probable highest and provide a good foundation for preparation of C6 aldehyde in the HPL reaction.In the study of extraction and purification of LOX, the defatted soybean meal was purified by ammonium sulfate fractionation, dialysis, and DEAE-Sepharose FF column chromatography, the specific activity was increased from 3.54U/mg to 105.7U/mg, purification fold of 29.9, the recovery is 30.9%. The study of LOX Characteristics shows that the optimal pH was pH9.0, a high level of activity was maintained at a lower temperature, with the double reciprocal Method, Km value was 80.6μmol / L, Vmax value was 54.2μmol / (L ? min).The influence of LOX catalytic reaction of the hydroperoxide conversion rate and the relationship of catalytic selectivity were determined by HPLC and LC-MS, so the optimal reaction conditions were obtained. In the reaction of linolenic acid as substrate, 0.025M of linolenic acid and 2.5U/ml the enzyme at pH9.0, 4℃, 2atm, 800rpm, 1.5h, the conversion rate was 83% and the purity was 87% under this conditions. In the reaction of Linoleic acid as substrate, 0.01M linoleic acid and 0.33U/ml enzymes at pH9.0, 4℃, 3atm, 800rpm,1.5h, the conversion rate was 81.3% and the purity was 97% under this conditions,Under the condition of the reaction with high substrate concentration,0.3M linolenic acid, 0.15% Triton X-100, 0.044gBHT and 20U/ml of LOX at pH9.0, 4℃, 2atm,1.5h,the conversion rate was 81.87% under the conditions. 0.3M linoleic acid, 0.2% Triton X-100, 0.08gBHT and 15U/ml of LOX at pH9.0, 4℃, 2atm, 1.5h, the conversion rate was 71.7% under the conditions.When in low substrate concentration, the conversion rate of pure enzyme was slightly higher than the crude enzyme as the substrate was linoleic acid in LOX reaction, and the conversion rate of the pure and crude reaction were hardly the same as the substrate was linolenic acid. However, in higher substrate concentration, whatever the substrate was, the conversion rate of pure enzyme was higher than crude enzyme in LOX reaction. It indicated that the conversion rate could be improved with the pure LOX in higher substrate concentration.In the low temperatures, hydroperoxide with ethanol preserved was more stable than those without ethanol.The study of the product of LOX showed that there was little oxoacid and hydroxy ketones besides hydroperoxide, in addition, other compounds need further investigated.