Immobilized glucose dehydrogenase for cofactor regeneration in heme-catalyzed demethylation and hydroxylation reactions

Glucose dehydrogenase (E.C. 1.1.1.47) from B. megaterium M 1286 was immobilized together with mutarotase (E.C. 5.1.3.3) on several organic carriers and by different methods. The storage stability of the enzyme at pH-values > 6 is slightly improved by immobilization and the pH-optimum is shifted from 8.3 to 8.0. Kinetic constants of the immobilized enzyme are: K′_M(NAD⁺) = 5.36 × 10^?4 mol/l K′_M(glucose) = 3.76 · 10^?2 mol/l and V′_max = 5.54 · 10^?5 mol/(l min g carrier) for the most active preparation (2.16 mg enzyme/g carrier). In reactor experiments the immobilized glucose dehydrogenase was used with glucose to regenerate NADPH in NADPH-dependent iron-III-protoporphyrin-IX-imidazole catalyzed hydroxylation and demethylation of model substrates of cytochrome P-450. The advantages of the coupling of both reactions with cofactor recycling are shown and discussed.