Site-specifically immobilized d-amino acid dehydrogenase for the synthesis of d-phenylalanine.

Abstract

Aromatic d-amino acids (d-AAs) have gained increasing attention as chiral building blocks, with biocatalytic procedures emerging as powerful methods for their asymmetric synthesis. d-Amino acid dehydrogenases (DAADH), developed by protein engineering from meso-diaminopimelate dehydrogenases, step out as highly efficient biocatalysts for the reductive amination-based production of d-AAs. Enzyme immobilization allows the recovery and reuse of biocatalysts, while also enhances their operational stability, which is essential for industrial applications. Since the immobilization of DAADHs have been less explored, we targeted to further progress within the covalent immobilization of DAADH from Ureibacillus thermosphaericus, by exploring its site-specific, covalent immobilization. The individual replacement of several surficial Ser residues to Cys at positions 2, 58, 92, 185, 192 and 317 of UtDAADH, allowed their site-specific immobilization onto the maleimide-functionalized Purolite® ECR8415F methacrylic support. The highest specific activity values, ~0.078 and ~0.083 U/mg provided by immobilization through Cys2 and Cys192, respectively, showed 2.1- and 2.2-fold higher values compared to the covalently, but non-specifically immobilized UtDAADH. The recyclability of the immobilized preparations was tested among 10 reductive amination-cycles of phenylpyruvate and based on the retained conversion and specific activities, UtDAADH immobilized through S2C was the best-performing biocatalyst, maintaining 65-70% conversion and 50% of the initial activity after the 10^th cycle. After the assessment of optimal enzyme/substrate ratio, the immobilized S2C UtDAADH was tested in three consecutive 200 mg-scale reaction, providing the enantiopure d-Phe with complete conversions and excellent >88% isolation yields, supporting its synthetic applicability.