Immobilization of peroxidase enzyme onto the porous silicon structure for enhancing its activity and stability

Abstract

In this work, a commercial peroxidase was immobilized onto porous silicon (PS) support functionalized with 3-aminopropyldiethoxysilane (APDES) and the performance of the obtained catalytic microreactor was studied. The immobilization steps were monitored and the activity of the immobilized enzyme in the PS pores was spectrophotometrically determined. The enzyme immobilization in porous silicon has demonstrated its potential as highly efficient enzymatic reactor. The effect of a polar organic solvent (acetonitrile) and the temperature (up to 50°C) on the activity and stability of the biocatalytic microreactor were studied. After 2-h incubation in organic solvent, the microreactor retained 80% of its initial activity in contrast to the system with free soluble peroxidase that lost 95% of its activity in the same period of time. Peroxidase immobilized into the spaces of the porous silicon support would be perspective for applications in treatments for environmental security such as removal of leached dye in textile industry or in treatment of different industrial effluents. The system can be also applied in the field of biomedicine.

Keywords: Immobilization; Microreactors; Peroxidase; Porous silicon.

Figure 1

Schematic diagram illustrating the general process from porous silicon functionalization to enzyme coupling. (a) Functionalization of oxidized porous support with ADPES. (b) Attachment of aldehyde group using glutaraldehyde. (c) Covalent attachment of peroxidase to the support through the formation of peptide bond between the aldehyde group and amino acids of the enzyme.

Figure 2

SEM observation of porous silicon structure fabricated, (a) cross section, (b) sample surface.

Figure 3

Shift in optical thickness (2nd) of the porous silicon structure after functionalization. The increase of the refractive index after the incubation in APDES and GTA results in a red shift in the reflectance peak, and hence, the corresponding change in optical thickness is observed.

Figure 4

Attenuated total reflectance (ATR) spectrum of PS structure with immobilized peroxidase taken after all the functionalization steps. FTIR analysis reveals some characteristic peaks of different functional group and peroxidase that has been infiltrated into the porous support.

Figure 5

First-order rate constant calculations from semi-logarithmic plot of residual activity of soluble and immobilized peroxidase during incubation (50°C).

Figure 6

First-order rate constant calculations from semi-logarithmic plot of residual activity of soluble and immobilized peroxidase during incubation (50% acetonitrile). The insert shows an amplification of immobilized enzyme profile.

Figure 7

First-order rate constant calculations from semi-logarithmic plot of residual activity of soluble and immobilized peroxidase with H ₂ O ₂ incubation.