An engineered <i>Phlebia radiata</i>manganese peroxidase: expression, refolding, purification and preliminary characterization

• 作者: Monday I. Akpanabiatu Usenobong F. Ufot
• 来源期刊: 美国分子生物学期刊(英文)
• 学科: 医学
• 关键词: Phlebia RADIATA PEROXIDASE Manganese PEROXIDASE OVER-EXPRESSION Recombinant
• 年，卷（期）: mgfzswxqkyw_2012,（4）
• 页码范围: 359-370
• 页数: 12页
• 分类号: R73

摘要

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 short-type 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 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 describes the spectral characterization of the wild-type and mutant rPr-MnP3 enzymes with are 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.