Abstract
An enzymatically active fungal manganese peroxidase (MnP) from Phanerochaete chrysosporium was synthesized in an in vitro coupled transcription–translation system. The synthesized MnP had the expected molecular mass (43,000 Da) and catalyzed the oxidation of 2,6-dimethoxyphenol (DMP) in the presence of hydrogen peroxide and Mn(II). A distal arginine residue (Arg 42) of the peroxide binding pocket and the potential N-glycosylation site (Asn 131) was site-directed mutagenized and corresponding mutant enzymes were also in vitro synthesized. Activities of the mutant enzymes towards 2,6-DMP were not significantly compromised although their dynamic characteristics were obviously different from the wild-type enzyme. The effect of the mutations was explained by using a computer-based three-dimensional modeling. These results demonstrated that in vitro expression of MnP provided a convenient and efficient system for characterization of fungal peroxidases.
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