Enhancement of Peroxidase Stability Against Oxidative Self-Inactivation by Co-immobilization with a Redox-Active Protein in Mesoporous Silicon and Silica Microparticles

Abstract

The study of the stability enhancement of a peroxidase immobilized onto mesoporous silicon/silica microparticles is presented. Peroxidases tend to get inactivated in the presence of hydrogen peroxide, their essential co-substrate, following an auto-inactivation mechanism. In order to minimize this inactivation, a second protein was co-immobilized to act as an electron acceptor and thus increase the stability against self-oxidation of peroxidase. Two heme proteins were immobilized into the microparticles: a fungal commercial peroxidase and cytochrome c from equine heart. Two types of biocatalysts were prepared: one with only covalently immobilized peroxidase (one-protein system) and another based on covalent co-immobilization of peroxidase and cytochrome c (two-protein system), both immobilized by using carbodiimide chemistry. The amount of immobilized protein was estimated spectrophotometrically, and the characterization of the biocatalyst support matrix was performed using Brunauer-Emmett-Teller (BET), scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDX), and Fourier transform infrared (FTIR) analyses. Stability studies show that co-immobilization with the two-protein system enhances the oxidative stability of peroxidase almost four times with respect to the one-protein system. Thermal stability analysis shows that the immobilization of peroxidase in derivatized porous silicon microparticles does not protect the protein from thermal denaturation, whereas biogenic silica microparticles confer significant thermal stabilization.

Keywords: Auto-inactivation; Microparticles; Peroxidase; Porous silica; Porous silicon.

Fig. 1

Pore size distribution of (black square) PSi and (black circle) BSiO₂ microparticles and the inset shows the nitrogen adsorption desorption isotherms of these two mesoporous materials

Scheme 1

Microparticles were treated with undecenoic acid to get carboxy-terminated microparticles. Microparticles were then incubated with peroxidase along with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS)

Scheme 2

Microparticles treated with 10-undecenoic acid and then incubated with cytochrome c along with EDC and NHS. These cytochrome-immobilized particles were washed with phosphate buffer and then incubated with peroxidase, EDC, and NHS

Fig. 2

SEM image. (a, d) Top view. (b, e) Surface view. (c) 2f cross sectional of PSi microparticles and BSiO₂ microparticles

Fig. 3

EDX results for PSi microparticles. a PSi microparticles, b cytochrome c immobilized to microparticles, and c peroxidase immobilized to microparticles

Fig. 4

EDX results for BSiO₂ microparticles. a BSiO₂ microparticles, b cytochrome c immobilized to microparticles, and c peroxidase immobilized to microparticles

Fig. 5

FTIR of one-protein and two-protein PSi-based biocatalysts

Fig. 6

FTIR of one-protein and two-protein BSiO₂-based biocatalysts

Fig. 7

Residual activity of soluble peroxidase and one-protein and two-protein biocatalysts during incubation at 50 °C

Fig. 8

Residual activity of one-protein and two-protein biocatalysts upon incubation with 1 mM H₂O₂