Mesoporous Silica Materials Labeled for Optical Oxygen Sensing and Their Application to Development of a Silica-Supported Oxidoreductase Biocatalyst

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 O-2 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 O-2 concentration profiles in solution and inside porous support to result exclusively from variable degrees of diffusion-caused limitation in the internal O-2 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 O-2-dependent oxidoreductases on porous silica material.