Stability of free and immobilised peroxidase in aqueous-organic solvents mixtures

The activity and stability of horseradish (Amoracia rusticana) peroxidase ( HR-P) free in solution and immobilised onto silica microparticles was studi ed in the presence of organic co-solvents. The effect of several hydrophilic organic solvents, namely dimethyl sulfoxi de, dimethylformamide, dioxan, acetonitrile and tetrahydrofuran, in the act ivity and stability of free HRP was studied. From the solvents tested, DMSO led to the highest activities and stabilities. After 2 h of incubation at 35 degreesC, the remaining activity of the enzyme in the presence of 30% of each solvent was less than 30%, with exception of DMSO for which the enzym e remained fully active. In order to increase stability, HRP was covalently immobilised onto silica microparticles. The half-life of the enzyme in buffer at 50 degreesC increa sed from 2 to 52 h when the enzyme was immobilised. The stability of both f ree and immobilised HRP was also studied at 50 degreesC in aqueous mixtures of 3.5, 20, 35 and 50% (v/v) DMSO. Free HR-P stability was not affected by the presence of 3.5 and 20% DMSO, but higher contents lead to a more prono unced deactivation. Immobilised HRP stability increased with DMSO content u p to 20%, decreasing for higher contents. The enzyme half-life increased mo re than 300% when changing from buffer to 20% DMSO. The deactivation of free HRP was modelled using the simple exponential deca y, and the deactivation of immobilised HRP was described by a two-step inac tivation model. (C) 2001 Elsevier Science B.V. All rights reserved.