Enzymatic Reduction of Nicotinamide Biomimetic Cofactors Using an Engineered Glucose Dehydrogenase: Providing a Regeneration System for Artificial Cofactors

The increasing demand for chiral compounds supports the development of enzymatic processes. Dehydrogenases are often the enzymes of choice due to their high enantioselectivity combined with broad substrate acceptance. However, their requirement on costly NAD(P)/H as cofactor has sparked interest in the development of biomimetic derivatives that are easy to synthesize and, therefore, less expensive. Until now, few reactions with biomimetics have been described and regeneration is limited to nonenzymatic means, which are not suitable for incorporation and in situ approaches. Herein, we describe a regeneration enzyme, glucose dehydrogenase from Sulfolobus solfataricus (SsGDH), and demonstrate its activity with different biomimetics with the structure nicotinamide ring-alkyl chain-phenyl ring. Subsequent enzyme engineering resulted in the double mutant SsGDH Ile192Thr/Val306Ile, which had a 10-fold higher activity with one of the biomimetics compared with the wild-type enzyme. Using this engineered variant in combination with an enoate reductase from Thermus scotoductus resulted in the first enzyme-coupled regeneration process for biomimetic cofactor without ribonucleotide or ribonucleotide analogue and full conversion of 10 mM 2-methylbut-2-enal with 1-phenethyl-1,4-dihydropyridine-3-carboxamide as cofactor.