Immobilized hydroxysteroid dehydrogenases for the transformation of steroids in water-organic solvent systems

Abstract

The hydroxysteroid dehydrogenases: β-HSDH, 20β-HSDH, and 3α-HSDH, were immobilized on CNBr-activated Sepharose. The effect of various immobilization conditions on the activity recovery and stability were examined. The presence of cofactor during the immobilization reaction increased the activity recovery (40–60% of the total) and also led to materials highly stable in the presence of organic solvents. For example, β-HSDH maintained 60% of its original activity two months after continuous use in the water–ethyl-acetate system. Kinetic experiments showed that the increase of the apparent K_m values is poor and demonstrated that the organic solvent behaves as a weak inhibitor (k_i > 0.2M) for the substrate. The immobilized enzymes lyophilized in the presence of sucrose had full activity restored even after several months storage at room temperature. Immobilized hydroxysteroid dehydrogenases were shown to be suitable for preparative transformation of steroids in water–organic solvent systems.