Biocatalysis of Glucose 2-Oxidase from Coriolus Versicolor at High Pressures Amin Karmali*, José P. Coelho , Denise P. Costa and Ana R. Barbosa, Centro de Investigação de Engenharia Química e Biotecnologia - DEQ, ISEL, Rua Conselheiro Emídio Navarro, 1950-062 Lisboa, Portugal. E-mail: akarmli@deq.isel.ipl.pt ; Fax: +351-218317267 Glucose 2-oxidase (pyranose oxidase, pyranose:oxygen-2-oxidoreductase, EC 1.1.3.10) from Coriolus versicolor catalyses the oxidation of D-.glucose at carbon 2 in the presence of molecular oxygen producing D-glucosone (2-keto-glucose and D-arabino-2-hexosulose) and hydrogen peroxide. This enzyme was used to convert D- glucose into D-glucosone at high pressures with compressed air in a modified commercial batch reactor. Several parameters affecting biocatalysis at high pressures were investigated as follows: pressure, enzyme concentration, glucose concentration, supercritical fluid and the presence of catalase. Glucose 2-oxidase was purified by immobilized metal affinity chromatography on epoxy-activated Sepharose 6B-IDA-Cu(II) column at pH 6.0. The conversion of D-glucose into D-glucosone was dependent on the pressure since an increase in the pressure with compressed air resulted in higher rates of conversion. On the other hand, the presence of catalase increased the rate of reaction which strongly suggests that hydrogen peroxide inhibited the rate of reaction. The rate of conversion of D-glucose into D- glucosone by glucose 2-oxidase in the presence of either nitrogen or supercritical CO 2 at 110 bar was very low compared with the use of compressed air at the same pressure. INTRODUCTION Enzymes are of great commercial interest because they have a number of applications in industry and Medicine such as textile, leather, detergent, starch, diagnostics, therapy and bioremediation. However, enzymes are not widely used in fine chemical industry mainly due to their poor stability. Glucose 2-oxidase (pyranose oxidase, pyranose:oxygen-2-oxidoreductase, EC 1.1.3.10) catalyses the oxidation of glucose at the carbon position 2 in the presence of oxygen producing D-glucosone and hydrogen peroxide (1). This enzyme is synthesized by white rot fungi such as Coriolus versicolor and Phanerochaete chrysosporium and plays an important role in lignin biodegradation (2). On the other hand, this enzyme is of great importance since the reaction product (D-glucosone) is an important precursor for biosynthesis of the antibiotic cortalcerone (3). The use of compressed air in this enzyme reaction would increase the rate of reaction since oxygen is one of the reaction substrates. However, high pressures also affect the enzyme structure altering its enzyme activity (4). Enzyme reactions can be carried out under high pressure or in supercritical fluids which is a new and promising field of enzyme engineering (5). High pressure is responsible for direct conformational changes in enzymes which affect their biological activities. Quaternary structure is very sensitive to pressure it is generally maintained by hydrophobic interactions which are rather weak interactions. In fact, moderate pressures have been found to favour the dissociation of oligomeric proteins such as lactate dehydrogenase. On the other hand, the secondary and tertiary structures are more resistant to pressure than the quaternary structure