Immobilization of d-amino acid dehydrogenase from Ureibacillus thermosphaericus

Several procedures were tested for the immobilization of the artificial d-selective amino acid dehydrogenase from Ureibacillus thermosphaericus (UtDAADH) and its co-immobilization with the NADPH–regenerating glucose dehydrogenase (GDH). Based on the conversions of the reductive amination of phenylpyruvate, recyclability, batch-to-batch reproducibility, and immobilization costs, DAADH covalently attached onto Purolite® ECR8415F or co-immobilized with GDH on polyethylenimine-coated agarose were selected for optimizations. The non-desired substrate/product adsorption occurring in case of the Purolite® support, was avoided by increasing the volume of linkers employed for the covalent fixation of the enzyme. The more convenient to prepare immobilization variant, DAADH adsorbed onto the Purolite® resin also proved to be applicable. This preparation, despite presenting significant drop of 58.8% of its specific activity over 10 reaction cycles, still provided similar conversions with the covalently immobilized variant. As third effective preparation, the DAADH–GDH co-immobilized system, showed no activity loss over 10 reaction cycles. In this case the additional co-immobilization of the NADP+ cofactor provided self-sufficient biocatalysts only for limited cycles, after >3 consecutive reactions the conversion dropped with ∼60%, due to cofactor leakage. The synthetic utility of the immobilized DAADH was demonstrated by the 100 mg-scale reductive amination of phenylpyruvate, obtaining d-phenylalanine with 86% yield.