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- W2031330779 abstract "We have optimized the conditions under which recombinant Mn peroxidase from the white-rot fungusPhanerochaete chrysosporiumcan be expressed inEscherichia coli.A bacterial expression vector for the cDNA of Mn peroxidase isozyme H4 (λMP1) was constructed (R. E. Whitwam, I. G. Gazarian, and M. Tien,Biochem. Biophys. Res. Commun.216, 1013–1017, 1995) whose expression inE. coliresults in the formation of catalytically inactive polypeptide which can be refolded to active enzyme. The refolded enzyme was purified to homogeneity. Refolding was most efficient in 2Murea, pH 8.0, and was absolutely dependent upon the presence of CaCl2, hemin, and oxidized glutathione. The recombinant enzyme had the same spectral and kinetic properties as the native fungal enzyme. TheKmof recombinant Mn peroxidase for substrates H2O2and the Mn2+/oxalate complex are 100 and 52 μM, respectively. Thekcatas measured by Mn3+/oxalate formation is 450 s−1. These are essentially the same values as seen with the native fungal enzyme. The rate of formation of compound I, the two-electron-oxidized state of the enzyme, is 4.0 × 106M−1s−1, identical to the rate of the native fungal Mn peroxidase. The reaction of compound I with Mn2+is too fast to measure at pH 4.5 in the recombinant Mn peroxidase. At a suboptimal pH of 2.5 a rate of 4.2 × 104M−1s−1is obtained for the recombinant enzyme. The reaction of compound II, the one-electron-oxidized state of the enzyme, with Mn2+/oxalate has aKdof 13 μMand a first-order rate constant of 230 s−1in the recombinant enzyme. These rates are essentially the same as those seen with the native fungal MnP. These results demonstrate that the bacterial expression of recombinant Mn peroxidase is a convenient and efficient system for the expression and characterization of Mn peroxidase." @default.
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- W2031330779 date "1996-09-01" @default.
- W2031330779 modified "2023-09-26" @default.
- W2031330779 title "Heterologous Expression and Reconstitution of Fungal Mn Peroxidase" @default.
- W2031330779 doi "https://doi.org/10.1006/abbi.1996.0413" @default.
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