Chemical Modification of Turnip Peroxidase with Methoxypolyethylene Glycol Enhances Activity and Stability for Phenol Removal Using the Immobilized Enzyme

Abstract

Peroxidase from turnip roots (TP) was isolated followed by modification with methoxypolyethylene glycol (MPEG). The catalytic activity of the modified TP (MTP) on ABTS increased 2.5 times after 80 min of reaction. MTP showed a KM similar value to that of TP, but a significantly greater kcat for ABTS oxidation, in aqueous buffer. Chemical modification produced an enhanced stability in organic solvents and increased thermal stability of about 4 times that of TP, in aqueous buffer at 70 degrees C. Circular dichroism showed that MPEG modification decreased TP alpha-helical structure from 26 to 16% and increased beta-turns from 26 to 34%, resulting in an enhanced conformational stability. The temperature at the midpoint of thermal denaturation (melting temperature) increased from 57 to 63 degrees C after modification. MTP was immobilized in alginate beads (IMTP) and tested for oxidative polymerization of concentrated phenolic synthetic solutions, achieving 17 effective contact cycles removing >65% phenols. IMTP may be useful for the development of an enzymatic process for wastewater effluent treatment.

Coupling Chemical Modification and Immobilization to Improve the Catalytic Performance of Enzymes

“…The catalytic activity of the modified enzyme increased 2.5 times. Chemical modification produced an enhanced stability in organic solvents and increased thermal stability about 4 times and the melting temperature from 57 to 63 °C 132. The modified enzyme was immobilized in alginate beads to have a reusable biocatalyst and tested for oxidative polymerization of concentrated phenolic synthetic solutions, achieving 17 effective cycles removing more than 65% of phenols.…”

Application Potential of Food Protein Modification

“…From a pharmaceutical viewpoint, PEGylation has been reported to enhance circulation life of bovine liver catalase in the blood of mice while the presence of PEG does not induce an immune response upon injection (Abuchowski et al, 1977). Modification of peroxidase from turnip was shown to enhance catalytic activity of the enzyme with increased stability in organic solvents as well as increased temperature resistance (Quintanilla-Guerrero et al, 2008). Similar results were found upon PEGylation of trichosanthin, which showed prolonged plasma half-life and reduced immunogenicity (He et al, 1999).…”

PEGylation and antioxidant effects of a human glutathione peroxidase 1 mutant

“…Such as PEGylated asparaginase (Oncaspar ® ) [ 7 ], PEGylated interferon α -2b (PEG-Intron ® ) [ 8 ], and PEGylated adenosine deaminase (Adagen ® ) [ 9 ]. The modification of proteins by the covalent conjugation of polyethylene glycol (PEG) shows great potential for enhancing enzyme stability [ 10 ], preventing effects due to temperature, pH, and proteolytic digestion [ 11 ], and improving pharmaceutical properties [ 12 , 13 ]. Proteins conjugated with PEG not only show reduced immunogenicity and renal clearance [ 14 ] but also exhibit improved half-time and stability in the blood [ 15 ] by preventing hydrolysis with proteolytic enzymes [ 16 ].…”