Cysteine Protease Inhibitors Produced by the Industrial Koji Mold, Aspergillus oryzae O-1018
T Yamada1, J Hiratake2, M Aikawa1
1a Research Institute, Gekkeikan Sake Co. Ltd.
Abstract:
Aspergillus oryzae O-1018 (FERM P-15834) separated from industrial koji for brewing sake was found to produce five papain-inhibitory compounds in the culture supernatant. The five isolated inhibitors were named CPI-1 to CPI-5, and their structures were elucidated by spectroscopic analyses and chemical degradation. We determined the structures of CPI-2, CPI-3 and CPI-4 as 4-amino-1-[[N- [(2S, 3S)-3-trans-carboxyoxiran-2-carbonyl]-L-isoleucyl] amino]butane, 5-amino-1-[[N-[(2S, 3S)-3-trans-carboxyoxiran-2-carbonyl]-L-isoleucyl]amino]pentane and N (8)- [N-[(2S, 3S)-3-trans-carboxyoxiran-2-carbonyl]-L-isoleu-cyl]spermidine, respectively. We also confirmed by a degradation experiment that CPI-1 consisted of L-trans-epoxysuccinic acid, L-tyrosine and spermidine, and that CPI-5 was composed of L-trans-epoxysuccinic acid, L-phenylalanine and spermidine. Although CPI-4 was identified as kojistatin A,(1)) the other CPIs seemed to be novel compounds. All CPIs were cysteine protease-specific inhibitors with appreciable selectivity toward cathepsin B and L. The inhibition potency of CPIs against cysteine proteases was as high as or higher than that of E-64. In particular, CPI-2, -3 and -4 were ten times more effective than E-64 toward cathepsin B and L, and CPI-1 and -5 were about 100 times more inhibitory than E-64 toward cathepsin L.
Insights
Five novel papain-inhibitory compounds, CPI-1 to CPI-5, were isolated from Aspergillus oryzae. These compounds exhibit potent cysteine protease inhibition, with some being significantly more effective than E-64.
Area of Science:
- Biochemistry
- Microbiology
- Enzymology
Background:
- Aspergillus oryzae is utilized in industrial sake brewing.
- Papain is a cysteine protease with various applications.
- The search for novel protease inhibitors is ongoing.
Purpose of the Study:
- To isolate and characterize papain-inhibitory compounds produced by Aspergillus oryzae O-1018.
- To elucidate the structures of these novel inhibitors.
- To evaluate their inhibitory activity against cysteine proteases.
Main Methods:
- Fermentation of Aspergillus oryzae O-1018.
- Isolation and purification of inhibitory compounds using chromatography.
- Structure elucidation via spectroscopic analyses (NMR, MS) and chemical degradation.
- Enzyme inhibition assays against cysteine proteases like cathepsin B and L.
Main Results:
- Five papain-inhibitory compounds, designated CPI-1 to CPI-5, were isolated.
- Structures of CPI-2, CPI-3, and CPI-4 were determined as novel compounds.
- CPI-1 and CPI-5 were identified as combinations of L-trans-epoxysuccinic acid, amino acids, and spermidine.
- All CPIs demonstrated specific inhibition of cysteine proteases, with CPI-1 and CPI-5 showing superior inhibition against cathepsin L compared to E-64.
Conclusions:
- Aspergillus oryzae O-1018 produces novel cysteine protease inhibitors.
- The identified CPIs possess significant inhibitory potential against cathepsin B and L.
- These compounds represent promising candidates for therapeutic or research applications targeting cysteine proteases.
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