Functional Expression and One-Step Protein Purification of Manganese Peroxidase 1 (rMnP1) from Phanerochaete chrysosporium Using the E. coli-Expression System

Abstract

Manganese peroxidases (MnP) from the white-rot fungi Phanerochaete chrysosporium catalyse the oxidation of Mn2+ to Mn3+, a strong oxidizer able to oxidize a wide variety of organic compounds. Different approaches have been used to unravel the enzymatic properties and potential applications of MnP. However, these efforts have been hampered by the limited production of native MnP by fungi. Heterologous expression of MnP has been achieved in both eukaryotic and prokaryotic expression systems, although with limited production and many disadvantages in the process. Here we described a novel molecular approach for the expression and purification of manganese peroxidase isoform 1 (MnP1) from P. chrysosporium using an E. coli-expression system. The proposed strategy involved the codon optimization and chemical synthesis of the MnP1 gene for optimised expression in the E. coli T7 shuffle host. Recombinant MnP1 (rMnP1) was expressed as a fusion protein, which was recovered from solubilised inclusion bodies. rMnP1 was purified from the fusion protein using intein-based protein purification techniques and a one-step affinity chromatography. The designated strategy allowed production of an active enzyme able to oxidize guaiacol or Mn2+.

Characterization of a Dye-Decolorizing Peroxidase from Irpex lacteus Expressed in Escherichia coli: An Enzyme with Wide Substrate Specificity Able to Transform Lignosulfonates

“…The I. lacteus DyP coding sequence was cloned into pET28a and expressed in E. coli BL21 (DE3) pLysS as described in Materials and Methods. Small scale fermentations showed recombinant protein production as inclusion bodies, as reported for other fungal hemoperoxidases [36,37]. To obtain an active enzyme, it was necessary to optimize an in vitro refolding protocol.…”

Textile Dye Biodecolorization by Manganese Peroxidase: A Review

“…For example, the synthetic MnP1 gene was cloned into the pTXB1 vector using Nde I/ Sap I restriction sites and delivered into Escherichia coli DH5α cells [ 87 ]. The overall process for the construction of pTXB1-MnP1 is summarized in the diagram depicted in Figure 4 .…”

Enzyme‐assisted bioremediation approach for synthetic dyes and polycyclic aromatic hydrocarbons degradation

“…Phanerochaete chrysosporium was the first white-rot fungus that was reported for having lignin-degrading peroxidases. Currently, all known white-rot fungus have this lignin-degrading peroxidase, and their capability of degrading lignin is also based on the availability of this enzyme [59,60]. MnP is extensively found in G. lucidum IBL-05 [55], Pleurotus ostreatus [61], C. unicolor BBP6 [56], as well as in Bjerkandera adusta strain CX-9 [62].…”