Biocatalysis of immobilized lipoxygenase and hydroperoxide lyase in organic solvent media

The secondary structure of commercially purified soybean lipoxygenase (LOX) type I-B as well as its immobilization and biocatalysis in organic solvent media (OSM) were investigated. The Fourier transform infrared (FT-IR) spectra of LOX obtained in chloroform, methanol and acetonitrile showed an absorption band at 1617 cm-1 indicative of significant protein aggregation, whereas spectra of lipoxygenase in hexane and octane exhibited substantially less aggregate formation. Variable-temperature infrared studies of lipoxygenase in D2O show that the predominately alpha-helical structure of the protein undergoes an irreversible transition to intermolecular beta-sheet at and above 65°C. The biocatalysis of free and immobilized (EupergitRTM C250L/EDA) LOXs was investigated in different mixtures of hexane and a selected cosolvent (95:5, v/v). The results showed a 1.5 and a 1.6-fold increase in the enzymatic activity of free and immobilized LOXs, respectively, using a mixture of hexane and 1,4-dioxane as compared to that in hexane. To determine the enzymatic production of hydroperoxides of linoleic acid in OSM, the xylenol orange (FOX) assay was optimized. An increase in the proportion of methanol from 0 to 75% in the FOX reagent resulted in a 93% increase in the molar absorption coefficients at 560 nm. Moreover, when perchloric acid was used, the source of ferrous ions and presence of denatured LOX had little effect on the sensitivity of the FOX assay whereas sensitivity decreased by 40 and 46%, respectively, with sulfuric acid. In addition, the immobilization of an enriched enzymatic extract of Penicillium camemberti, containing LOX and hydroperoxide lyase (HPL) activities, and its biocatalysis in OSM were investigated. The highest immobilization efficiency (173.9 and 694.4% for LOX and HPL, respectively) was obtained with unmodified EupergitRTMC250L. The increase in thermal stability of both LOX and HPL activities were obtained by the immobilization of the enriched enzymatic extract on EupergitRTMC250L. The thermal inactivation of LOX did not follow first order kinetic behavior but the one of HPL did. Moreover, the biocatalysis of LOXi and HPLi in OSM resulted in an increase in their enzymatic activity. The use of 60% iso-octane (v/v) in the reaction medium increased the HPLi activity by more than 5 fold as compared to that in the aqueous medium. The specific activity of LOXi was increased to a maximum of 7.73 nmol HPODs/mg immobilized protein/min with an increase in tetradecane concentration up to 20% (v/v) in the ternary micellar system. Similarly, the addition of tetradecane in the reaction medium increased further the HPLi activity by 17% as compared to that in 60% iso-octane medium without tetradecane.