Kinetic and redox properties of MnP II, a major manganese peroxidase isoenzyme from Panus tigrinus CBS 577.79.

摘要

A manganese peroxidase (MnP) isoenzymefrom Panus tigrinus CBS 577.79 was produced in a benchtop stirred-tank reactor and purified to apparent homogeneity. The purification scheme involving ultrafiltration, affinity chromatography on concanavalin–A Sepharose, and gel filtration led to a purified MnP, termed ‘‘MnP II,’’ with a specific activity of 288 IU mg-1 protein and a final yield of 22%. The enzyme turned out to be a monomeric protein with molecular mass of 50.5 kDa, pI of 4.07, and an extent of N-glycosylation of about 5.3% of the high-mannose type. The temperature and pH optima for the formation of malonate manganic chelates were 45°C and 5.5, respectively. MnP II proved to be poorly thermostable at 50 and 60°C, with half-lives of 11 min and 105 s, respectively. Km values for H2O2 and Mn2+ were 16 and124 lM, respectively. Although MnP II was able to oxidize veratryl alcohol and to catalyze the Mn2+ -independentoxidation of several phenols, it cannot be assigned to the versatile peroxidase family. As opposed to versatile peroxidaseoxidation, veratryl alcohol oxidation required the simultaneous presence of H2O2 and Mn2+; in addition, low turnover numbers and Km values higher than 300 lMcharacterized the Mn2+ -independent oxidation of substituted phenols. Kinetic properties and the substrate specificityof the enzyme markedly differed from those reported for MnP isoenzymes produced by the reference strain P. tigrinus 8/18. To our knowledge, this study reports for the first time a thorough electrochemical characterization of a MnP from this fungus.