Dissecting Physical and Biochemical Factors of Catalytic Effectiveness in Immobilized D-Amino Acid Oxidase by Real-Time Sensing of O2 Availability Inside Porous Carriers

Juan Manuel Bolivar Bolivar, Sabine Schelch, Torsten Mayr, Bernd Nidetzky

Research output: Contribution to journalArticle

Abstract

D‐Amino acid oxidase (DAAO) presents a paragon for effective use of biocatalytic O2‐dependent transformations in fine‐chemical and pharmaceutical synthesis at the industrial scale. Solid‐supported DAAO immobilizates are applied to continuous processing, but their activity and stability are often inadequate. Targeted immobilization development is restricted by insufficient knowledge of physical and biochemical factors governing the performance of heterogeneous DAAO catalysts. We have applied real‐time optical sensing of the O2 availability in luminescence‐labeled porous Sepabeads and ReliSorb carriers to quantify diffusional restrictions in DAAO immobilizates differing in the mode of enzyme attachment to the solid surface. We show that noncovalent oriented immobilization of DAAO (from Trigonopsis variabilis) resulted in high retention of the original enzyme activity (≥60 %), whereas covalent multipoint fixation caused massive (up to 90 %) activity loss. Depletion of O2 inside the solid immobilizates became limiting for enzyme catalytic effectiveness at activity loadings as low as 5 units gcarrier−1. Slow pore diffusion was principally responsible for the observed large mass‐transfer resistance, and this provides a main starting point for process intensification.

D‐Amino acid oxidase (DAAO) catalyzes the oxidative deamination of R‐configured α‐amino acids with absolute enantioselectivity but very broad substrate specificity.1 Flavin adenine dinucleotide (FAD) in the oxidized form is the enzyme’s cofactor for hydrogen abstraction from the α C atom of the substrate. The resulting α‐imino acid is usually left to rapid hydrolysis in water, which thus eventually yields an α‐keto acid product.1a, 1b Reduced FAD reacts with O2 to restore the cofactor’s original redox state to give H2O2 as the second product.1e The paramount example of the use of DAAO in industrial biotransformation development is the two‐step chemoenzymatic conversion of cephalosporin C into 7‐aminocephalosporanic acid (7‐ACA), which is the essential precursor for the synthesis of various cephem antibiotics (Scheme 1 a). The industrial 7‐ACA process pioneered the application of O2‐dependent biocatalysis on a large (multiton per year) manufacturing scale.2 Dynamic kinetic resolution of racemic (nonnatural) α‐amino acids is another application of DAAO in industrial fine‐chemical synthesis.1d The commercialized process involves interception of the enzymatically formed α‐imino acid by completely unselective chemical reduction. Multiple rounds of oxidation and reduction eventually result in a highly enriched (S)‐α‐amino acid product (Scheme 1 b).1d
Original languageEnglish
Pages (from-to)981-986
JournalChemCatChem
Volume6
Issue number4
DOIs
Publication statusPublished - 2014

Fields of Expertise

  • Human- & Biotechnology

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