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

Abstract

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.

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.…”

“…O 2 availability in supernatant (∼90 % air saturation at atmospheric pressure) was regularly checked with the oxygen microoptode. At certain times, a homogeneous sample was withdrawn and the concentration of 2-keto-4-(methylthio)butyric acid was determined 16 . At the end of reaction, the enzyme immobilizate was recovered by centrifugation, and it was used again for new rounds of conversion.…”

“…16 Figure 3 shows CLSM images recorded from supports loaded with a relatively high amount of enzyme (2000 U/g_support). At this loading, clear signs of hindered immobilization were already noted for most of the silica materials, as indicated in Figure 4A.…”

“…Enzyme chimeras harboring Z basic2 are efficiently immobilized on plain silica-based materials. 16,45 The immobilization is highly selective, most likely because it appears to occur in a well-defined orientation via the Z basic2 module. It is stable under operation, yet readily reversible.…”

“…It is stable under operation, yet readily reversible. 16,45 High protein loading (> 50 mg/g carrier) is possible in principle, but this depends strongly on the material properties of the OMSM used. Unfortunately, these material properties are often not ideal, or even unsuitable, for enzyme immobilization.…”