An engineered Phlebia radiata manganese peroxidase: expression, refolding, purification and preliminary characterization
American Journal of Molecular Biology
Manganese peroxidases (MnPs) are interesting enzymes in protein engineering, aimed at maximizing industrial bioprocesses such as lignin degradation and biofuel production. cDNA of the secreted shorttype of MnP from Phlebia radiata (Pr-MnP3) has been successfully engineered and amplified by polymerase chain reaction (PCR). Five mutant genes (E40H, E44H, E40H/E44H, D186H and D186N) of recombinant Phlebia radiata MnP3 (rPr-MnP3) were generated. The wild-type and the mutant genes were expressed in
... were expressed in Escherichia coli (W3110 strain) and the resultant body proteins were lysed, purified and refolded into active enzymes. 6% -7% recovery of pure and fully active rPr-MnP3 for wild-type and mutants were obtained and the availability of rPr-MnP3 enzymes will greatly facilitate its structure-function relationships studies. rPr-MnP3 mass was characterised using SDS-PAGE and MALDI-TOF mass spectrometry. Molecular weight of both the wild-type and mutant rPr-MnP3 enzymes was approximately 36 kDa. This paper describes the spectral characterization of the wild-type and mutant rPr-MnP3 enzymes with very close similarities; substantially high spin haem enzymes. Therefore we report the engineering, cloning, expression, refolding/activation of MnP3 genes and preliminary characterization of the wild-type and mutant Phlebia radiata MnP3 enzymes.