IMMOBILIZATION OF MANGANESE PEROXIDASE FROM LENTINULA EDODES ON AZLACTONE-FUNCTIONAL POLYMERS AND GENERATION OF MN3-POLYMER COMPLEX( BY THEENZYME)

Manganese peroxidase (MnP) purified from Lentinula edodes was covalent ly immobilized on 3M's azlactone-functional copolymer, 3M Emphaze (TM) AB1 Biosupport Medium. Tethered MnP is capable of generating Mn3+ fro m Mn2+ and H2O2. Mn3+, properly chelated, can be used as a nonspecific oxidant of organopollutants. A variety of conditions designed to maxi mize coupling efficiency while maintaining Mn3+-generating catalytic a ctivity were tested. Biochemical characteristics of the MnP enzyme, in cluding amino acid composition, pH and temperature stability, and conc entration of its Mn2+ substrate, influenced chemical conditions necess ary for the coupling reaction. The physical parameters of immobilizati on reaction time, protein concentration, ionic conditions, pH, and tem perature 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 c ontaining 1.0M sodium sulfate and 1.0 mM Mn2+. Increasing coupling rea ction temperature also improved coupling efficiency. A synthesis of th ese optimized immobilizations yielded MnP coupling efficiencies of 40- 50% with 35% of the coupled protein retaining enzymatic activity. Resu lts of MnP immobilizations on nonporous azlactone-functional dispersio n polymers more hydrophobic than Emphaze are also reported, and coupli ng efficiencies >65% with 100% of the coupled enzyme active have been measured.