Stabilization of horseradish peroxidase in aqueous-organic media by immobilization onto cellulose using a cellulose-binding-domain

Abstract

A fused protein consisting of a cellulose-binding domain (CBD) and horseradish peroxidase (HRP) was bound to cellulose beads and evaluated in aqueous-organic solvent systems. The CBD–HRP fusion protein containing two different functionalities, a catalytic domain and a binding domain, preserved both capabilities in this non-conventional environment. A six-fold increase in the half-life of the enzyme in buffer resulted from immobilization onto cellulose via CBD. The immobilized enzyme was also more stable than the native enzyme in increasing concentrations of acetone (0–92%). There was a general decrease in activity as the solvent concentration in the mixture increased (in all solvent types: THF, acetone, acetonitrile and ethanol). However, the immobilized enzyme was at all times more active than the soluble enzyme forms. The thermostability of the enzyme in buffer, at 40–60 °C, was also improved by immobilization. The soluble CBD–HRP fusion protein exhibited greater stability (both to organic solvents and temperature), but lower activity, in comparison with the native HRP. This work demonstrates for the first time the use of a cellulose-bound CBD-enzyme as a catalyst in aqueous-organic solvent media.