Journal of Molecular Catalysis B: Enzymatic 15 (2001) 147–153 Stability of free and immobilised peroxidase in aqueous–organic solvents mixtures Ana M. Azevedo, Duarte M.F. Prazeres, Joaquim M.S. Cabral, Lu´ ıs P. Fonseca ∗ Centro de Engenharia Biológica e Qu´ ımica, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal Received 28 November 2000; received in revised form 6 March 2001; accepted 6 March 2001 Abstract The activity and stability of horseradish (Amoracia rusticana) peroxidase (HRP) free in solution and immobilised onto silica microparticles was studied in the presence of organic co-solvents. The effect of several hydrophilic organic solvents, namely dimethyl sulfoxide, dimethylformamide, dioxan, acetonitrile and tetrahydrofuran, in the activity 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 ◦ C, 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 enzyme 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 ◦ C increased from 2 to 52 h when the enzyme was immobilised. The stability of both free and immobilised HRP was also studied at 50 ◦ C in aqueous mixtures of 3.5, 20, 35 and 50% (v/v) DMSO. Free HRP stability was not affected by the presence of 3.5 and 20% DMSO, but higher contents lead to a more pronounced deactivation. Immobilised HRP stability increased with DMSO content up to 20%, decreasing for higher contents. The enzyme half-life increased more than 300% when changing from buffer to 20% DMSO. The deactivation of free HRP was modelled using the simple exponential decay, and the deactivation of immobilised HRP was described by a two-step inactivation model. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Horseradish peroxidase; Immobilisation; Silica; Dimethyl sulfoxide; Thermostability 1. Introduction The use of aqueous mixtures of organic co-solvents for conducting enzymatic reactions has become highly promising in biotechnology processes. By total or partial replacement of water as the reaction medium with an organic solvent, hydrophobic substrates can be more efficiently converted, hydrophobic products can be produced with higher yields, and the thermo- ∗ Corresponding author. Tel.: +351-218-419139; fax: +351-218-419062. E-mail address: lfonseca@alfa.ist.utl.pt (L.P. Fonseca). dynamic equilibrium of hydrolytic reactions can be shifted to the synthetic way [1,2]. Moreover, some enzymes are often more stable in water-poor envi- ronments than in water [3]. Nevertheless, increasing the concentration of an organic solvent in the reac- tion medium can lead to the inactivation of enzymes due to reversible changes in the protein structure, while a more prolonged incubation leads to the ir- reversible inactivation of the enzyme [4,5]. There are several approaches to delay both reversible and irreversible inactivation. One of them is to choose a solvent, which disturbs the catalytically active con- formation of the protein to a lesser extent. Another 1381-1177/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII:S1381-1177(01)00017-0