Porous silica materials make great supports for heterogeneous catalysis with immobilized enzymes; however, direct functionalization of their surface through stable attachment of enzymes, reporter molecules, or both is a difficult problem. Overcoming that is necessary for practical implementation. Here, we integrate the development of luminophor-doped oxygen-sensing silica materials with a modular strategy of enzyme immobilization to demonstrate generally applicable design of an oxygen-dependent biocatalyst on a porous silica support. Zbasic2, a highly positively charged silica-binding module of about 7 kDa size, was fused to d-amino acid oxidase, and the resulting chimeric protein was tethered noncovalently via Zbasic2 in defined orientation and in a highly selective manner on silica. The enzyme supports used differed in overall shape and size as well as in internal pore structure. A confocal laser scanning microscopy (CLSM) analysis that employed the oxidase’s flavin cofactor as the fluorescent reporter group showed a homogeneous internal protein distribution in all supports used. Ru-based organometallic luminophor was adsorbed tightly onto the silica supports, thus enabling internal optical sensing of the O2 available to the enzymatic reaction. Optimization of the surface labeling regarding homogeneous luminophor distribution was guided, and its efficacy was verified by CLSM. Mesostructured silica surpassed controlled pore glass by ≥10-fold in terms of immobilized enzyme effectiveness at high loading of oxidase activity. The effect was shown from detailed comparison of the time-resolved O2 concentration profiles in solution and inside porous support to result exclusively from variable degrees of diffusion-caused limitation in the internal O2 availability. Enzyme immobilized on mesostructured silica approached perfection of a heterogeneous biocatalyst in being almost as effective as the free enzyme (assayed in oxidative deamination of d-methionine), thus emphasizing the large benefit of targeted mass transfer intensification, through proper choice of support parameters, in the development of immobilizates of O2-dependent oxidoreductases on porous silica material.

[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

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Sialo-oligosaccharides are important products of emerging biotechnology for complex carbohydrates as nutritional ingredients. Cascade bio-catalysis is central to the development of sialo-oligosaccharide production systems, based on isolated enzymes or whole cells. Multienzyme transformations have been established for sialooligosaccharide synthesis from expedient substrates, but systematic engineering analysis for the optimization of such transformations is lacking. Here, we show a mathematical modeling-guided approach to 3ʹ-sialyllactose (3SL) synthesis from N-acetyl-D-neuraminic acid (Neu5Ac) and lactose in the presence of cytidine 5ʹ-triphosphate, via the reactions of cytidine 5ʹ-monophosphate-Neu5Ac synthetase and α2,3-sialyltransferase. The Neu5Ac was synthesized in situ from N-acetyl-Dmannosamine using the reversible reaction with pyruvate by Neu5Ac lyase or the effectively irreversible reaction with phosphoenolpyruvate by Neu5Ac synthase.We show through comprehensive time-course study by experiment and modeling that, due to kinetic rather than thermodynamic advantages of the synthase reaction, the 3SL yield was increased (up to 75%; 10.4 g/L) and the initial productivity doubled (15 g/L/h), compared with synthesis based on the lyase reaction. We further show model-based optimization to minimize the total loading of protein (saving: up to 43%) while maintaining a suitable ratio of the individual enzyme activities to achieve 3SL target yield (61%-75%; 7-10 g/L) and overall productivity (3-5 g/L/h).Collectively, our results reveal the principal factors of enzyme cascade efficiency for 3SL synthesis and highlight the important role of engineering analysis to make multienzyme-catalyzed transformations fit for oligosaccharide production.

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