Study On The Issues In Catalysis Of Soybean Lipoxygenase And The Synthesis And Application Of Hydroperoxide Product

Lipoxygenase (LOX) can catalyze the oxidation of polyunsaturated fatty acids with cis,cis-1,4-pentadiene to synthesize the corresponding hydroperoxides (HPOD) of conjugated (trans, cis)-dienes. In this paper, some key issues in the catalysis of LOX and the synthesis of hydroperoxide product, for example, the control of exogenous metal ions on the LOX catalyzed reaction process, the substitution of Cu2+ with the iron in the active site of LOX, the connection between LOX activity and its conformation were studied. Besides, using soymeal as the catalyst, selecting linoleic acid, ethyl linoleate and soybean oil as the substrate, and ultrasonic irradiation was introduced into the lipoxygenation process, a green and efficient way to produce hydroperoxide product was given with satisfactory yield. The main points were as follows:The effect of exogenous metal ions on the conformation and catalytic activity of LOX was observed. The addition of exogenous Cu2+,Fe3+ and Fe2+ in LOX solvent strengthened the absorbance band between 300-360nm in UV spectrum of LOX significantly. And they could quench the fluorescence excitation spectra of LOX. A quantitative proof of replacing iron (II) inside LOX by exogenous Cu2+ was provided, simply using UV-Vis spectrometry with two indicators p-carboxylantipyrylazo and 9-(4-carboxyphenyl)-2,3,7- trihydroxyl- 6-fluorine.The inactivation and secondary structure variation of soybean lipoxygenase during heating process was studied by FT-IR and CD technology. Thermal inactivation of LOX could be described by a first order kinetic model (LOX) activity was determined by spectrophotometrically measured changes in residual activity), activation energy Ea was further calculated to be 217 kJ/mol. The quantitative secondary structures of LOX at different temperatures were obtained by CD technology and FT-IR spectra processed by Omnic6.0 and Peakfit4.12. During 50-65 oC, the variation range of secondary structures of LOX was not as significant as that of thermal inactivation. Both CD and FT-IR analysis showed that the change of secondary structure of LOX was not the main reason for the loss of its activity.With simultaneous enzyme releasing and lipoxygenation, the catalytic performance of soymeal to catalyze the hydroperoxidation reaction with ethanol as co-solvent was evaluated, using linoleic acid, ethyl linoleate and soybean oil as the substrate. The influences of various parameters relating to the hydroperoxide yield, including co-solvent, pH, temperature, and dosage of soymeal and soybean oil were investigated. The optimal conditions for soybean oil were 1.5g soybean oil in 7.5 mL phosphate buffer solution of pH 6 with 0.75 mL ethanol as co-solvent reacting for 3 h at 20 oC, and the yield reached 61%, which is much higher than the previously reported. Soymeal has much better catalytic performance than crude LOX (30%). The application of soymeal not only reduces the number of steps by coupling extraction of LOX and oxidation reaction, but also increased the total yield. The employment of ultrasonic irradiation during the lipoxygenation process accelerated the enzymatic reaction and reduced the catalyst soymeal amount significantly (by 75%). In the conditions of 10 g soybean oil, 1g soymeal and 1 mL ethanol dissolved in 7.5 mL pH6 phosphate buffer at 15 oC, the ultrasonic assisted hydroperoxidation of soybean oil reached equilibrium after 60 min with the yield of 61%. So a facile, cheap and practical biotransformation catalyzed by soymeal was provided for the synthesis of lipid hydroperoxide with satisfactory yield. The use of renewable materials such as soymeal and soybean oil approaches a more realistic scenario for the industrial scale.Linoleic acid hydroperoxide (LA-HPOD-Na) is a green multifunctional surfactant having both bleaching and washing performance. Comparing with sodium perborate and sodium percarbonate, HPOD-Na performed best on bleaching methylene blue but worst on chlorophyl (the bleaching rate reached 30% and 7% at 30 oC in 30 min, respectively). Two commercial detergents Diaopai soap powder and Aomiao soap powder were selected as promising formula that had good synergism with HPOD-Na. The surface tension of hydroxy linoleic acid (HOD-Na) was 30.56 mN/m, and it had better emulsifying, foaming and dispersion than linoleic acid.