Sunday, August 12, 2012
Columbia Hall, Terrace Level (Washington Hilton)
Chloroperoxidase (CPO) from Caldariomyces fumago is one of the most attractive peroxidases due to its catalytic versatility. In this work, we explored the covalent immobilization of CPO in Eupergit C, a commercial acrylic-based support. Several strategies were tested. In the first one, CPO was immobilized with glutaraldehyde to hexanediamine-derivatized Eupergit C. A load of 25-37 mgCPO/gsupport was attained, however the enzyme lost 95% activity. In the second strategy, lysine residues were derivatized with dithiopropanoic acid to generate thiol groups, unique functional groups in the surface of the enzyme, with the aim of avoiding multipoint attachment and active site blockage. The modified enzyme was attached to maleimide-activated Eupergit C. The immobilized CPO retained 40% activity and a load of 0.04-0.09 mg CPO/g support was observed. In the third strategy, CPO was adsorbed on Eupergit C activated with diazonium ions. We achieved a load of 14-34 mgCPO/gsupport with 100% of retained activity. Desorption of the enzyme from the support was observed in aqueous solution, however we have found relative stability in a binary solution comprising tert-butanol and phosphate buffer (80:20 v/v). During the first 6 hours half of CPO activity was lost, while residual activity was maintained for the next 30 hours. This is the first time that successful immobilization of CPO on Eupergit C is reported.