Immobilization of horseradish peroxidase as crosslinked enzyme aggregates (CLEAs)

Abstract

Herein we report the successful preparation of catalytically active crosslinked enzyme aggregates (CLEAs®) of peroxidase (HRP, EC 1.11.1.7) from horseradish roots (Armoracia rusticana or Cochlearia armoracia) using various aggregation agents, followed by crosslinking with glutaraldehyde. The percent activity recovery of the HRP-CLEAs was 83% of that of the native enzyme. The CLEAs were prepared in the presence of egg albumin (37mg/mL) and pentaethylenehexamine (PEHA; 10mM). The albumin played a significant role in stabilizing the CLEA particles, and the PEHA was necessary to obtain fully crosslinked HRP aggregates. Because HRP possesses only six Lys (lysine) residues, the addition of PEHA increases the number of free amino groups (–NH2) on the outer surface of the enzyme, thus facilitating the crosslinking. A potential application of HRP aggregates is envisaged for coupled oxidation reactions with cholesterol oxidase in the biosensor field.