Fourier transform infrared study of lipoxygenase conformation in organic solvent media.

The secondary structure of commercially purified soybean lipoxygenase (EC 1.13.11.12) was investigated in selected monophasic organic solvents, including chloroform, methanol, acetonitrile, hexane, and octane. The Fourier transform infrared (FT-IR) spectra of the enzyme 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 D(2)O show that the predominately alpha-helical structure of the protein undergoes an irreversible transition to intermolecular beta-sheet at and above 65 degrees C. Chemical imaging technology employing an FT-IR spectrometer equipped with an infrared microscope and a focal-plane array detector was used to examine the changes in the secondary structure of lipoxygenase at the water-hexane interface in the presence and absence of substrate. The secondary structure of lipoxygenase at the hexane-water interface was comparable to that of the structure of lipoxygenase in D(2)O after exposure of lipoxygenase solution to hexane.

Stability of immobilized soybean lipoxygenase in selected organic solvent media.

The immobilization and biocatalysis of commercially purified soybean lipoxygenase (LOX) type I-B (EC 1.13.11.12) were investigated in organic solvent media. The results showed that the highest immobilization efficiencies of LOX, 30.6 and 29.3%, were obtained with DEAE-cellulose and modified Eupergit C250L supports, respectively. The biocatalysis of free and immobilized (Eupergit 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 increase in the activity of free and immobilized LOXs, respectively, using a mixture of hexane and 1,4-dioxane compared with that in hexane alone; however, cosolvents, including 2-octanone, 2-heptanone, 2-butanone, and cyclohexanone, displayed an inhibitory effect on LOX activity. In the mixture of 1,4-dioxane and hexane, LOX activity was dependent on the cosolvent concentration, which was increased with 1,4-dioxane up to 5% (v/v). The threshold 1,4-dioxane concentration (C50) and the incubation period (T50) at which 50% of the maximal enzyme activity was obtained for the free and immobilized LOXs were 6.7 and 8.9% (v/v) and 9.1 and 17.0 min, respectively.