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Characterization of an aspartic proteinase of Mucor pusillus expressed in Aspergillus oryzae
K Murakami1, J Aikawa, S Horinouchi
1Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Japan.
Abstract:
The aspartic proteinase (MPP) gene from the zygomycete fungus Mucor pusillus was introduced into an ascomycete fungus, Aspergillus oryzae, by protoplast transformation using the nitrate reductase (niaD) gene as the selective marker. Southern blot analysis indicated that the MPP gene was integrated into the resident niaD locus at a copy number of 1-2. MPP secreted by the recombinant A. oryzae was correctly processed but was more highly glycosylated than that produced in the original M. pusillus strain. Treatment with endo-beta-N-acetylglucosaminidase H and analysis of the carbohydrate composition of the secreted MPP revealed that the extra glycosylation of the MPP secreted by the recombinant A. oryzae was due to altered processing of mannose residues. The extra glycosylation of MPP affected its enzyme properties including its milk-clotting and proteolytic activities.
Insights
Genetic engineering introduced the Mucor pusillus aspartic proteinase (MPP) gene into Aspergillus oryzae. The resulting recombinant fungus produced MPP with altered glycosylation, affecting its milk-clotting and proteolytic activities.
Area of Science:
- Molecular biology
- Biotechnology
- Enzymology
Background:
- The aspartic proteinase (MPP) is an important enzyme produced by Mucor pusillus.
- Genetic engineering offers a method to produce valuable enzymes in alternative host organisms.
- Understanding enzyme processing and activity in recombinant systems is crucial for biotechnological applications.
Purpose of the Study:
- To introduce the Mucor pusillus aspartic proteinase (MPP) gene into Aspergillus oryzae.
- To characterize the processing, glycosylation, and enzymatic activity of MPP produced in the recombinant A. oryzae.
- To investigate the impact of altered glycosylation on MPP's functional properties.
Main Methods:
- Protoplast transformation of Aspergillus oryzae with the MPP gene from Mucor pusillus, using the niaD gene as a selective marker.
- Southern blot analysis to confirm gene integration and copy number.
- Biochemical analysis of secreted MPP, including endo-beta-N-acetylglucosaminidase H treatment and carbohydrate composition analysis.
- Assays to evaluate milk-clotting and proteolytic activities of the recombinant MPP.
Main Results:
- Successful integration of the MPP gene into the A. oryzae genome.
- Recombinant A. oryzae secreted correctly processed MPP, but with increased glycosylation compared to the native strain.
- Altered mannose residue processing in A. oryzae led to the enhanced glycosylation of MPP.
- The heightened glycosylation of MPP impacted its milk-clotting and proteolytic enzyme activities.
Conclusions:
- Aspergillus oryzae can be engineered to produce Mucor pusillus aspartic proteinase (MPP).
- Host-specific glycosylation differences in A. oryzae alter MPP's enzymatic properties.
- This study provides insights into heterologous protein expression and enzyme modification in fungal systems.