IMMOBILIZATION OF MANGANESE PEROXIDASE FROM LENTINULA EDODES ON AZLACTONE-FUNCTIONAL POLYMERS AND GENERATION OF MN3+ BY THE ENZYME-POLYMER COMPLEX

Manganese peroxidase (MnP) purified from Lentinula edodes was covalently immobilized on 3M's azlactone-functional copolymer, 3M Emphaze (TM) AB1 Biosupport Medium. Tethered MnP is capable of generating Mn3+ from Mn2+ and H2O2. Mn3+, properly chelated, can be used as a nonspecific oxidant of organopollutants. A variety of conditions designed to maximize coupling efficiency while maintaining Mn3+-generating catalytic activity were tested. Biochemical characteristics of the MnP enzyme, including amino acid composition, pH and temperature stability, and concentration of its Mn2+ substrate, influenced chemical conditions necessary for the coupling reaction. The physical parameters of immobilization reaction time, protein concentration, ionic conditions, pH, and temperature were examined. Results of these experiments indicated maximum coupling efficiency and enzyme activity were achieved by immobilizing at MnP concentrations <2 mg/mL for at least 2 h using pH 7.0 buffer containing 1.0M sodium sulfate and 1.0 mM Mn2+. Increasing coupling reaction temperature also improved coupling efficiency. A synthesis of these optimized immobilizations yielded MnP coupling efficiencies of 40-50% with 35% of the coupled protein retaining enzymatic activity. Results of MnP immobilizations on nonporous azlactone-functional dispersion polymers more hydrophobic than Emphaze are also reported, and coupling efficiencies >65% with 100% of the coupled enzyme active have been measured.