Immobilized horseradish peroxidase on discs of polyvinyl alcohol-glutaraldehyde coated with polyaniline.

Samantha Salomão Caramori,Luiz Bezerra De Carvalho Junior,Kátia Flávia Fernandes

doi:10.1100/2012/129706

Abstract

Discs of network polyvinyl alcohol-glutaraldehyde (PVAG) were synthesized and coated with polyaniline (PANI) using glutaraldehyde as a chemical arm (PVAG-PANIG-HRP disc). The best conditions for the immobilization were established as about 1.0 mg mL−1 of protein, for 60 min and pH 5.5. The soluble enzyme lost all of its activity after incubation at 70°C for 15 min, whereas the PVAG-PANIG-HRP disc retained about half of the initial activity for pyrogallol. The same PVAG-PANIG-HRP disc was used consecutively three times without any activity lossbut presented 25% of the initial activity after the 7th use. PVAG-PANIG-HRP disc retained approximately 80% and 60% of its initial activity after 60 and 80 days of storage, respectively. Resorcinol, m-cresol, catechol, pyrogallol, α-naphthol, βnaphthol, and 4, 4′-diaminodiphenyl benzidine were efficiently oxidized by the PVAG-PANIG-HRP disc (from about 70% to 90%), and it was less efficient towards aniline, phenol, and 2-nitrosonaphthol.

Highlights

Peroxidases (Enzyme Commission number 1.11.1.7) are a huge family of heme-containing enzymes that catalyze oxidation and reduction reactions of large families of substrates.Their broad substrate specificity, polyfunctionality, and availability from different sources allow their application in various biotechnological processes
The soluble enzyme lost all of its activity after incubation at 70◦C for 15 min, whereas the polyvinyl alcohol-glutaraldehyde (PVAG)-PANIG-Horseradish peroxidase (HRP) disc retained about half of the initial activity for pyrogallol
Further evidences for the higher thermal stability for the PVAG-PANIG-HRP can be seen in Figure 5 which showed that the soluble enzyme activity decreased faster than the immobilized preparation when both were incubated at 50◦C and 70◦C

Summary

Introduction

Peroxidases (Enzyme Commission number 1.11.1.7) are a huge family of heme-containing enzymes that catalyze oxidation and reduction reactions of large families of substrates. Peroxidases have a variety of biotechnology application due to this catalytic mechanism They are capable of oxidizing many compounds bearing the functional group R-N=N-R (azo-dyes), in which R and R can be either aryl or alkyl groups. Attempts to overcome the separation step were conducted by our research group, culminating in the preparation of PET-PANIG composite as strips with immobilized HRP, which could be manually removed from reaction medium. This material presented low protein loading [15]. Some features affecting the immobilization procedure and properties of this derivative were investigated

Materials and Methods

Results and Discussion

Conclusions