SELECTIVE OXIDATION - STABILIZATION BY MULTIPOINT ATTACHMENT OF FERREDOXIN NADP(+) REDUCTASE, AN INTERESTING COFACTOR RECYCLING ENZYME

Ferredoxin-NADP(+) reductase (FNR, EC 1.18.1.2) is an enzyme that is able to catalyse the oxidation of NADPH + H+. A strategy to prepare industrial derivatives of this enzyme for use as an 'NADP' regenerating enzyme in oxidizing reactions is presented. The strategy is based on a strictly controlled process of multipoint covalent attachment between the enzyme, via its amino groups, and a pre-existing solid activated with a monolayer of simple aldehyde groups linked by a space-arm of moderate length to the surface of the support. Controlling the variables which may have an influence in the multi-interaction process, we have prepared a number of enzyme derivatives with very different activity/stability properties. The 'optimum derivative' was found to be much more stable than its corresponding soluble enzyme under all the denaturation conditions assayed (high temperatures, extreme pH, organic solvents, etc.). Because of the excellent properties of this enzyme derivative, we can regenerate NADP(+) by using molecular oxygen directly as the oxidizing agent under a wide range of conditions. Coupling this oxidative system to other NADP-dependent redox enzymes, we should be able to develop a very specific and selective oxidative procedure under very mild oxidizing conditions.