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Studies on the production of fungal peroxidases in Aspergillus niger
A Conesa1, C A van den Hondel, P J Punt
1Department of Molecular Genetics and Gene Technology, TNO Nutrition and Food Research Institute, 3704 HE Zeist, The Netherlands.
Abstract:
To get insight into the limiting factors existing for the efficient production of fungal peroxidase in filamentous fungi, the expression of the Phanerochaete chrysosporium lignin peroxidase H8 (lipA) and manganese peroxidase (MnP) H4 (mnp1) genes in Aspergillus niger has been studied. For this purpose, a protease-deficient A. niger strain and different expression cassettes have been used. Northern blotting experiments indicated high steady-state mRNA levels for the recombinant genes. Manganese peroxidase was secreted into the culture medium as an active protein. The recombinant protein showed specific activity and a spectrum profile similar to those of the native enzyme, was correctly processed at its N terminus, and had a slightly lower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant MnP production could be increased up to 100 mg/liter upon hemoglobin supplementation of the culture medium. Lignin peroxidase was also secreted into the extracellular medium, although the protein was not active, presumably due to incorrect processing of the secreted enzyme. Expression of the lipA and mnp1 genes fused to the A. niger glucoamylase gene did not result in improved production yields.
Insights
Researchers explored fungal peroxidase production in Aspergillus niger, finding that manganese peroxidase (MnP) can be efficiently produced and secreted as an active enzyme. Lignin peroxidase (LiP) expression, however, resulted in inactive protein, highlighting processing challenges.
Area of Science:
- Biotechnology
- Molecular Biology
- Enzyme Engineering
Background:
- Filamentous fungi are crucial for producing enzymes like fungal peroxidases.
- Understanding limitations in enzyme production is key for industrial applications.
- Phanerochaete chrysosporium peroxidases (lignin peroxidase and manganese peroxidase) are of significant interest.
Purpose of the Study:
- To investigate the expression of Phanerochaete chrysosporium lignin peroxidase (lipA) and manganese peroxidase (mnp1) genes in Aspergillus niger.
- To identify limiting factors for efficient fungal peroxidase production.
- To optimize the production of active fungal peroxidases.
Main Methods:
- Utilized a protease-deficient Aspergillus niger strain for gene expression studies.
- Employed different expression cassettes for recombinant gene integration.
- Analyzed gene expression using Northern blotting.
- Assessed enzyme activity, N-terminal processing, and SDS-PAGE for secreted proteins.
- Investigated the effect of hemoglobin supplementation and gene fusion strategies.
Main Results:
- High steady-state mRNA levels were observed for both lipA and mnp1 genes.
- Active manganese peroxidase (MnP) was successfully secreted into the culture medium with properties similar to the native enzyme.
- Recombinant MnP production reached up to 100 mg/liter with hemoglobin supplementation.
- Secreted lignin peroxidase (LiP) was not active, suggesting incorrect processing.
- Fusion of genes to the Aspergillus niger glucoamylase gene did not improve production yields.
Conclusions:
- Aspergillus niger can efficiently produce active manganese peroxidase (MnP).
- Challenges in protein processing limit the production of active lignin peroxidase (LiP) in this system.
- Hemoglobin supplementation is a viable strategy to enhance MnP yields.
- Further research is needed to overcome processing issues for LiP production.