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Functional Expression and One-Step Protein Purification of Manganese Peroxidase 1 (rMnP1) from Phanerochaete
Angel De La Cruz Pech-Canul1, Javier Carrillo-Campos2, María de Lourdes Ballinas-Casarrubias2
1CONACyT-Faculty of Chemical Sciences, Autonomous University of Chihuahua, Campus II, Chihuahua 31125, Mexico.
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+.
Insights
Researchers developed a new method to produce manganese peroxidase (MnP) in E. coli. This approach enhances the production of active MnP1, a key enzyme for various applications.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Manganese peroxidases (MnP) from Phanerochaete chrysosporium are crucial oxidoreductive enzymes.
- Limited native production of MnP hinders research and application development.
- Previous heterologous expression systems faced challenges with low yield and process complexity.
Purpose of the Study:
- To develop an efficient molecular strategy for expressing and purifying manganese peroxidase isoform 1 (MnP1) from P. chrysosporium.
- To overcome limitations associated with native enzyme production and existing heterologous expression systems.
- To obtain an active and purified recombinant MnP1 (rMnP1) for further studies.
Main Methods:
- Codon optimization and chemical synthesis of the MnP1 gene for E. coli expression.
- Expression of MnP1 as a fusion protein in an E. coli T7 shuffle host.
- Purification of rMnP1 from inclusion bodies using intein-based techniques and affinity chromatography.
Main Results:
- Successful expression of recombinant MnP1 (rMnP1) as a fusion protein in E. coli.
- Efficient recovery and purification of active rMnP1.
- Demonstrated enzymatic activity of purified rMnP1 in oxidizing guaiacol and Mn2+.
Conclusions:
- The developed molecular strategy enables optimized expression and purification of active MnP1 in E. coli.
- This method provides a viable alternative for producing sufficient quantities of MnP for research and potential industrial applications.
- The purified rMnP1 exhibits functional activity, confirming the efficacy of the expression and purification strategy.
