Studied On Soybean Lipoxygenase Catalysis Under Aerobic Condition

Lipoxygenase (LOX) contains a nonheme iron in its catalytic center. It can catalyze the oxidation of polyunsaturated fatty acids containing cis,cis-1,4-pentadiene moiety to the corresponding hydroperoxides (HPOD) of conjugated (trans, cis)-dienes, which are important intermediates for synthesizing drugs and chemicals. Soybean lipoxygenase was chosen as a model in this paper, the optimal reaction conditions improved by dimethyl formamide (DMF), relaxing of substrate inhibition and product inhibition, and the interaction between exogenous Cu(II) and Fe(II)-LOX were investigated. The main points are as follows:The addition of DMF can not only overcome the higher viscosity resulted from higher substrate level, improve the substrate solubility and enhance the coexistence of substrate with enzyme, but also increase the yield of HPOD. At first, with single factor method the optimal reaction conditions were obtained;then the reaction conditions were further improved with pure linoleic acid (LA) as substrateby using orthogonal design . The final reaction conditions were as follows: pH 9, 10℃, 15 mL(O₂)/min, stirring linear velocity 5.02m/s, φ (DMF) =4 %, ρ (sodium citrate) =1 %, [LA]= 90¹00 g/L, [E]= 4.74×10⁵ U/mL, [antioxidant]= 0.02 g/L. Under the final condition, with fresh soybean produced from northeast China as enzyme source, [LA]= 90 g/L, up to 100% HPOD was achieved after reacted 90 mins. With HPLC analysis, HPOD includes two isomers, i.e. 13-HPOD and 9-HPOD with a ratio of 1.9 to l(mol/mol).The substrate inhibition was observed during the hydroperoxidation of LA catalyzed by LOX at substrate concentrations higher than 0.075 mmol/L. DMF (logP=-0.81) can not only enhance the substrate concentration to 232 mmol/L, but also increase the HPOD yield from 38.93% to 66.09%. Lyophilization test showed that DMF with substrate didn't do harm to LOX and the enzymatic activity. DMF served as either an activator at low DMF level or an inhibitor at high DMF level, which was indicated by the activation constant K_a , the inhibition constant K_i and the effect of DMF with substrate on enzymatic activity. The substrate inhibition constant K_ss at high DMF level was increased by 1000⁵600 folds compared to the control, which implied that the relaxation of substrate inhibition and competitive inhibition of LOX in DMF mediated system released the substrate inhibition greatly together . The maximum K_ss and K_i obtained at 5% DMF means that the maximum relaxation of substrate inhibition and the minimum competitive inhibition of LOX in DMF is appeared simultaneously.In the system the hydroperoxidation of LA catalyzed by LOX was improved by adding6% of DMF, the mechanism of electrolyte influencing LOX catalysis overall at high LA level should keep the following three key points in one rather than explaining only by the traditional theory of two substrate-binding sites competition. The three key points were, (1) adjusting the competitive adsorption of substrate between the two substrate-binding sites;(2) activating LOX in commonness and in individual caused by the characteristics of electrolytes;and (3) minor-modulating the binding status of LOX-LA-DMF. The effect of electrolyte on this catalysis responded to (1) or (2) was a monotonous increased function in respect of electrolyte level, but the one in answer to (3) showed a bell-shape. The apparent random phenomena caused by electrolytes type and level could be successfully explained by the integrating mechanism blending the above three factors. There was an optimal dosage of electrolyte in the LOX catalysis since the influence of electrolyte on the reaction was complex.There are two kind of effect of the product HPOD ( at a concentration of 14.8 nmol/L) on LOX. (1) HPOD's competitive inhibition on LOX at low substrate level (20.08 umol/L). (2) HPOD's activation on LOX at high substrate level (120.50 umol/L), relaxing the substrate inhibition.The addition of exogenous Cu( II) in LOX solvent, so does Fe(III) or LA-HPOD, caused the absorbance band between 300-400 nm in UV spectrum of soybean lipoxygenase strengthened. 9.449 umol/L Cu(II) has interacted with 16.10 umol/L LOX for 5 mins, the free Cu(II) in the solvent was determined by reagent p-chloroantipyrylazo and the exchanging ratio was 7.08% of LOX(mol/mol), the free Fe( II) was determined by the reagent 9-(4-carboxyphenyl)-2,3,7-trihydroxyl-6- fluorine and the exchanging ratio was 3.48% of LOX(mol/mol). All the above showed there existed the exchange between Cu( II) and LOX-Fe( II), in another word, it is possible for Fe( II) in the LOX to exchanged by other exogenous iron under proper conditions.