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

Abstract

[a] d-Amino acid oxidase (DAAO) presents a paragon for effective use of biocatalytic O 2 -dependent transformations in fine-chemical and pharmaceutical synthesis at the industrial scale. Solidsupported 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 O 2 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 O 2 inside the solid immobilizates became limiting for enzyme catalytic effectiveness at activity loadings as low as 5 units g carrier À1

Advanced characterization of immobilized enzymes as heterogeneous biocatalysts

“…Mid-IR spectroscopy [164,165] Raman spectroscopy Wide range of carriers Global reaction rates of stirred suspension of immobilized enzymes Immersed probes were used [157] [172,173] been applied [27, for biocatalyst screening and characterization as well as for study and optimization of the reactor operation. Table 5 presents a summary of the immobilized enzyme system studied, with a focus on applied aspects.…”

“…The quantification of spaced averaged intraparticle oxygen concentrations in porous polymethacrylate enzyme carriers was accomplished by labeling the enzyme carrier with an O 2 -sensitive luminophore and application of the phase modulation technique. Formation of a large gradient between O 2 concentrations in bulk solvent and the internal environment of the carrier was detected, clearly indicating limitations in the supply of O 2 co-substrate into the solid support [172,173]. In-operando determination of oxygen gradients between homogeneous liquid phase and internal catalytic environment was performed during determination of catalytic activity of heterogeneous oxidases as biocatalysts.…”

Oriented Coimmobilization of Oxidase and Catalase on Tailor-Made Ordered Mesoporous Silica

“…15,16 We have shown by experiment of earlier work that reaction-caused depletion of the O 2 substrate within the solid support can in fact become limiting for E obs of an immobilized oxidase, most notably under conditions when the E bound is high. 15,60 A useful approach to attenuate diffusional restrictions at high E bound is to optimize parameters of the carrier's geometry, specifically the particle size. Behavior of the PSSP immobilizates is exemplary in this regard ( Figure 5B).…”

Biosilica and bioinspired silica studied by solid-state NMR

“…In operando characterization of the catalyst (i.e. : in realistic conditions of applications of the biocatalyst) have been carried out by applying diverse analytical methods directly within the solid support: examples include optochemical sensing [68][69][70][71] as well as spectroscopic imaging and electrochemical sensing (see table 5 in reference [51]). These methods yield direct handle on reagents' local concentration, gradients and diffusion properties with both temporal and spatial information.…”

“…The introduction of NMR of sedimented solutes [69][70][71] has lifted the needs for crystallization in the case of soluble proteins, yielding access to soluble but non-crystallizing targets such as PEGylated proteins [130]. However, as seen by NMR, biomaterials are complex entities: the protein and the inorganic matrix interplay to define physicochemical characteristics of the composite, in terms of mobility, hydration, etc.…”