Evaluation of different immobilization strategies to prepare an industrial biocatalyst of formate dehydrogenase from Candida boidinii

Different immobilization strategies have been issued to prepare a stable and active biocatalyst of the formate dehydrogenase from Candida boidinii: immobilization on glutaraldehyde, epoxy, amino-epoxy, glyoxyl or treatment of enzymes adsorbed on aminated supports with glutaraldehyde. The best results in terms of stability were achieved using amino-epoxy supports (by a 12-fold factor compared to soluble and BrCN immobilized enzyme) and glyoxyl agarose supports (by a 150-fold factor), although in both cases activity recovery was just over 15%. The use of milder conditions permitted to improve the recovered activity, but decreased in a parallel way the enzyme stability, until being almost identical to the soluble enzyme. Alternatively, ionic adsorption of the enzyme was assayed. Supports coated with PEI permitted to keep full activity of the enzyme, permitted a very strong adsorption and the reutilization of the support after enzyme inactivation. Moreover, all the immobilization techniques prevent enzyme inactivation under strong stirring, necessary to keep the pH value and eliminate the CO₂ formed during the reaction. In this way, a battery of solutions to immobilize this enzyme has been proposed, fulfilling the different requirements that the use of this NADH recycling enzyme may have, depending on the particular reaction.