Stabilization of horseradish peroxidase by covalent conjugation with dextran aldehyde against temperature and pH changes

Melda Altikatoglu 1 , Candan Arioz 2 , Yeliz Basaran 2 , and Huriye Kuzu 2
  • 1 Faculty of Arts and Sciences, Department of Chemistry, Yildiz Technical University, Davutpasa Campus, 34210, Esenler, Istanbul, Turkey
  • 2 Faculty of Chemical and Metallurgical Engineering, Department of Bioengineering, Yildiz Technical University, Davutpasa Campus, 34210, Esenler, Istanbul, Turkey

Abstract

Stabilization of Horseradish Peroxidase (HRP; EC 1.11.1.7) against temperature and pH via the formation of the conjugates obtained by multipoint covalent bonding of dextran aldehyde (DA) to the enzyme were studied. Hence, three different molar weighted dextrans (17.5 kD, 75 kD, 188 kD) were covalently bonded to purified enzyme with different molar ratios (nHRP/nDA 20/1, 10/1, 1/1, 1/5, 1/10, 1/15, 1/20). The thermal stabilities of the obtained conjugates were evaluated with the activities determined at different temperatures (25, 30, 35, 40, 50, 60, 70, 80°C) applying 60 minutes incubation time. Conjugates formed were characterized by gel-permeation chromatography (GPC) and fluorescence techniques. The conjugate synthesized using dextran 75 kDa with nHRP/nDA 1/10 molar ratio showed better thermal stability than other conjugates and purified enzyme at pH 7. This conjugate also has wider activity pH range than purified enzyme. In addition, mentioned conjugate at pH 7 had very long storage lifetime compared to purified enzyme at +4°C and room temperature; which is considered a favorable feature for usage in practice.

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