linoleic-acid and 2-2-4-trimethylpentane

Lipoxygenase (LOX) is an enzyme that regioselectively introduces the hydroperoxide functionality into polyunsaturated fatty acids, such as linoleic acid (LA). Hydroperoxide derivatives of polyunsaturated fatty acids are of interest because they can serve as important intermediates in the synthesis of chemical and pharmaceutical compounds. In this study, LOX was immobilized in dispersed phyllosilicate layers that were cross-linked with silicate polymers formed by the hydrolysis of tetramethyl orthosilicates. The effects of substrate concentration, reaction temperature and solvent participation were studied on the oxidation of LA by LOX. The temperature optimum for the oxidation of LA by immobilized LOX was 25 degrees C and values of Km and Vmax for this reaction were 1.7 mM and 0.023 micromol/min respectively. Enzymic activity was stimulated by the addition of 10% (v/v) iso-octane to the reaction mixture. The immobilized LOX preparation showed a degree of substrate preference that demonstrated that 1,3-dilinolein was a better substrate than LA in the oxidation reaction, followed in order by 1-monolinolein, methyl oleate and trilinolein. In general, LOX immobilized in cross-linked phyllosilicates retained the physical and chemical characteristics of free LOX.

Topics: Diglycerides; Drug Compounding; Enzymes, Immobilized; Glycine max; Kinetics; Linoleic Acid; Lipoxygenase; Octanes; Plant Proteins; Silicates; Substrate Specificity; Temperature

The effective pH of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) reverse micelles (pHrm), containing buffers of different pH (pHst) and various amounts of linoleic acid, was studied within the range of compositions used to study the activity of soybean lipoxygenase in reverse micelles. Significant shifts of pHrm versus pHst were observed for the solutions of relatively higher pHst, dependent on linoleic acid and buffer concentrations. The effect diminished as pHst became closer to 7. When low-ionic-strength buffers were added to AOT solutions in isooctane, a significant buffering effect of linoleic acid in reverse micelles was observed. Solubilization of > 3 mM linoleic acid in micellar solutions containing 25 mM buffers gave the observed pHrm values almost independent of pHst. This effect diminished with the ionic strength of the buffering solution, but did not vanish even at 200 mM buffer. The observed effects result from the balance between ionization of linoleic acid and its partition between the water pool and the micellar interface. The enzymic activity of soybean lipoxygenase in the AOT reverse micellar solutions of the determined pHrm values was also studied. A significant reduction of the kinetics of the enzymic activity was observed, for all studied reverse micellar solutions. Changes of pHrm, caused by the presence of acidic substrate (linoleic acid) do not explain the observed reduction of activity directly through the effect on the enzyme. Due to unfavourable partition of the substrate between the microphases present in the systems, enhanced by reduction of pH at higher total concentrations of linoleic acid, the saturation of the enzyme with the substrate was not observed in the system and is difficult to attain experimentally in reverse micelles. A shift of the lipoxygenase activity/pHrm profile but negligible shift of the activity/pHst profile, with respect to aqueous buffer solutions, were observed. This indicates that either the information given by pH indicators used does not reveal the true pH of the enzyme in these reverse micelles, or that the sample of the enzyme is pH-insensitive over a broader pH range than results from the relationship observed for linoleic acid in aqueous solutions. The latter conclusion is consistent with the data on lipoxygenase activity towards linoleyl sulfate in aqueous solutions [Bild, G. S., Ramadoss, C. S. & Axelrod, B. (1977) Lipids 12, 732-735]. Conditions for measuring pH-independent lipoxygenase activ

Topics: Dioctyl Sulfosuccinic Acid; Glycine max; Hydrogen-Ion Concentration; Kinetics; Linoleic Acid; Linoleic Acids; Liposomes; Lipoxygenase; Micelles; Octanes; Succinates