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

Authors

  • Dr. Juan M. Bolivar,

    1. Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, 8010 Graz (Austria)
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  • Sabine Schelch,

    1. Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, 8010 Graz (Austria)
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  • Prof. Dr. Torsten Mayr,

    1. Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology, Stremayrgasse 9, 8010 Graz (Austria)
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  • Prof. Dr. Bernd Nidetzky

    Corresponding author
    1. Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, 8010 Graz (Austria)
    • Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, 8010 Graz (Austria)

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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.

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