Immobilization of a lipoxygenase in silica gels for application in aqueous media

The encapsulation of soybean lipoxygenase-1 (LOX-1) in silica gels and its application in an aqueous medium, were studied. The main silica precursor was tetramethoxysilane (TMOS) but the introduction of hydrophobic SiCH3 groups brought with methyltrintethoxysilane (MTMS) was evaluated. Other sod-gel synthesis parameters investigated comprised partial or complete drying by evaporation and CO2 supercritical drying. The influence on LOX-1 activity of the various chemicals with which the enzyme was in contact during encapsulation (acetone, methanol, polyvinyl alcohol), as well as the temperature and pH, were examined. The activity of free and encapsulated LOX-1 was assayed on the oxygenation reaction of linoleic acid by dioxygen from air dissolved in aqueous medium, in a UV-vis spectrophotometer. With free LOX-1, the reaction advancement could be followed in continuous in the spectrophotometer. With the gels, in a first approach, the conversion was simply determined after 15 min reaction after filtration of the liquid, to discriminate between active and inactive gels. For the most interesting gels, the kinetics were then assessed by continuous recording in the UV spectrophotometer, after placing a small piece of gel (approximate to 15 mg) directly in the cell. The best gels had an activity approximate to 30% of free LOX. The present studies, supplemented by characterization of the gels texture and structure, respectively by nitrogen adsorption and Si-29 MAS NMR, showed that drying a gel before use in aqueous media was detrimental to the activity. This effect is due to a contraction of the gel network which occurs when a dry aerogel sample is dipped in water after drying. Hence gels containing LOX-1 enzyme must not be dried but kept in water impregnated state, for optimum use. (c) 2006 Elsevier B.V. All rights reserved.