Bolívar Bolívar, Juan ManuelSchelch, SabineMayr, TorstenNidetzky, Bernd2024-01-092024-01-092015ACS Catal. 2015, 5, 10, 5984–5993 Publication Date:August 28, 2015 https://doi.org/10.1021/acscatal.5b016012155-543510.1021/acscatal.5b01601https://hdl.handle.net/20.500.14352/91995Porous 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.engAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/journal articlehttps://doi.org/10.1021/acscatal.5b01601open access546577.1BiocatalysisEnzyme immobilizationFusion proteinIntraparticle oxygen gradientOptical sensingOxygen-dependent oxidationsSilica binding moduleSilica materialsIngeniería químicaBiotecnologíaQuímica industrialQuímica física (Química)Química analítica (Química)2303 Química Inorgánica2307 Química Física2302 Bioquímica3302 Tecnología Bioquímica3303 Ingeniería y Tecnología Químicas