An engineered Phlebia radiata manganese peroxidase: Expression, refolding, purification and preliminary characterization

Manganese peroxidases (MnPs) are interesting en-zymes in protein engineering, aimed at maximizing industrial bioprocesses such as lignin degradation and biofuel production. cDNA of the secreted short-type of MnP from Phlebia radiata (Pr-MnP3) has been successfully engineered and amplified by poly-merase 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 Escherichia coli (W3110 strain) and the resultant body proteins were lysed, purified and re-folded 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 en-zymes will greatly facilitate its structure-function re-lationships studies. rPr-MnP3 mass was characterised using SDS-PAGE and MALDI-TOF mass spectrome-try. Molecular weight of both the wild-type and mu-tant 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, clon-ing, expression, refolding/activation of MnP3 genes and preliminary characterization of the wild-type and mutant Phlebia radiata MnP3 enzymes