Isolation and characterization of an extracellular antimicrobial protein from Aspergillus oryzae.
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.
Park
Seong-Cheol
SC
Research Center for Proteineous Materials (RCPM), Chosun University, Gwangju, 501-759, Korea.
Yoo
Nae Choon
NC
Kim
Jin-Young
JY
Park
Hae Kyun
HK
Chae
Byung Jo
BJ
Shin
Song Yub
SY
Cheong
Hyeonsook
H
Park
Yoonkyung
Y
Hahm
Kyung-Soo
KS
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D000935:Antifungal Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D001236:Aspergillus oryzae / Q000737:chemistry* / Q000378:metabolism; D001419:Bacteria / Q000187:drug effects; D005110:Extracellular Space / Q000737:chemistry* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D005658:Fungi / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D016415:Sequence Alignment
D000900:Anti-Bacterial Agents; D000935:Antifungal Agents; D005656:Fungal Proteins