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Published on: February 15, 2019
Isolation and characterization of an extracellular antimicrobial protein from Aspergillus oryzae
Seong-Cheol Park1, Nae Choon Yoo, Jin-Young Kim
1Research Center for Proteineous Materials (RCPM), Chosun University, Gwangju, 501-759, Korea.
Abstract:
A 17 kDa antimicrobial protein was isolated from growth medium containing the filamentous fungus Aspergillus oryzae by extracting the supernatants from the culture media, ion exchange chromatography on CM-sepharose, and C18 reverse-phase high-performance liquid chromatography. This antimicrobial protein, which we considered to be an extracellular antimicrobial protein from A. oryzae (exAP-AO17), possessed antimicrobial activity but lacked hemolytic activity. The exAP-AO17 protein strongly inhibited pathogenic microbial strains, including pathogenic fungi, Fusarium moniliform var. subglutinans and Colletotrichum coccodes, and showed antibacterial activity against bacteria, including E. coli O157 and Staphylococcus aureus. To confirm that the protein acts as a regulation factor for extracellular secretion, we examined growth under varying conditions of N sources, C sources, ions, ambient pH, and stress. Various culture conditions were found to induce characteristic changes in the expression of protein synthesis as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Highly basic polypeptides were regulated by suppressing the ambient pH under acidic conditions and strongly induced under alkaline conditions, thus confirming that pH regulation is physiologically relevant. The expression of exAP-AO17 was upregulated by heat shock upon growth in the presence of NaCl. Automated Edman degradation showed that the N-terminal sequence of exAP-AO17 was NH 2-GLPGPAGAVGFAGKDQNM-. ExAP-AO17 showed partial sequence homology with a collagen belonging to the animal source. These results suggest that exAP-AO17 is an excellent candidate as a lead compound for the development of novel oral or other types of anti-infective agents.
Insights
Researchers isolated a novel antimicrobial protein, exAP-AO17, from Aspergillus oryzae. This protein effectively inhibits pathogenic fungi and bacteria without causing hemolysis, showing potential for new anti-infective drug development.
Area of Science:
- Microbiology
- Biochemistry
- Mycology
Background:
- Filamentous fungi like Aspergillus oryzae are sources of bioactive compounds.
- Antimicrobial proteins are crucial for host defense and potential therapeutic agents.
- The need for novel anti-infective agents is driven by increasing antimicrobial resistance.
Purpose of the Study:
- To isolate and characterize a novel antimicrobial protein from Aspergillus oryzae.
- To evaluate the antimicrobial spectrum and hemolytic activity of the isolated protein.
- To investigate the regulation of the protein's expression under various environmental conditions.
Main Methods:
- Isolation and purification using chromatography techniques (ion exchange, reverse-phase HPLC).
- Antimicrobial activity assays against pathogenic fungi and bacteria.
- Hemolytic activity assays.
- Analysis of protein expression under different culture conditions (pH, N/C sources, stress).
- N-terminal sequencing by automated Edman degradation.
Main Results:
- A 17 kDa extracellular antimicrobial protein (exAP-AO17) was isolated.
- exAP-AO17 exhibited potent antimicrobial activity against Fusarium moniliform, Colletotrichum coccodes, E. coli O157, and Staphylococcus aureus.
- The protein showed no hemolytic activity.
- exAP-AO17 expression was regulated by ambient pH and induced by heat shock and NaCl.
- N-terminal sequencing revealed partial homology to animal collagen.
Conclusions:
- exAP-AO17 is a novel, non-hemolytic antimicrobial protein from Aspergillus oryzae.
- Its broad-spectrum antimicrobial activity and unique regulation suggest therapeutic potential.
- exAP-AO17 is a promising candidate for developing new anti-infective agents.