Abstract
The efficiency of enzymes in organic solvents is determined by the catalytic parameters, which mostly are changed in comparison with those in aqueous solutions, as well as by reversible and irreversible denaturation of the enzyme protein. The interplay of these processes was studied on soluble and immobilized glucoamylase and invertase in mixtures of buffer and several water-miscible solvents such as methanol, glycerol, dimethylformamide, and dioxane. A comparison of the catalytic activity in 50 or 30% (v/v) solvents with the extent of irreversible inactivation during the time of assay showed that the solvents act on the catalytic parameters of the enzymes and their conformational stability in different ways. Irreversible inactivation is especially strong in dimethylformamide, whereas glycerol does not induce denaturation but strongly reduces the catalytic efficiency. Striking effects were observed in dioxane, where high stability to irreversible denaturation but strong reduction of catalytic power were observed. In all solvents K m values were more affected than V max values, suggesting that reversible denaturation plays an inferior role in activity losses. Similar effects of the individual solvents were also observed after immobilization of the enzymes by covalent binding to a macroporous aminomethyl polystyrene-divinylbenzene copolymer or a macroporous aminopropyl silica. In all cases, however, immobilized enzymes showed better long-term stability than the soluble enzymes.
Published Version
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