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Fujieda N.,Osaka University | Murata M.,Osaka University | Yabuta S.,Osaka University | Ikeda T.,Osaka University | And 5 more authors.
ChemBioChem | Year: 2012

The pro form of melB tyrosinase from the melB gene of Aspergillus oryzae was over-produced from E. coli and formed a homodimer that exhibited the spectral features of met-tyrosinase. In the presence of NH 2OH (reductant), the proenzyme bound dioxygen to give a stable (μ-η 2:η 2-peroxo)dicopper(II) species (oxy form), thus indicating that the pro form tyrosinase can function as an oxygen carrier or storage protein like hemocyanin. The pro form tyrosinase itself showed no catalytic activity toward external substrates, but proteolytic digestion with trypsin activated it to induce tyrosinase activity. Mass spectroscopy analyses, mutagenesis experiments, and colorimetry assays have demonstrated that the tryptic digestion induced cleavage of the C-terminal domain (Glu458-Ala616), although the dimeric structure of the enzyme was retained. The structural changes induced by proteolytic digestion might open the entrance to the enzyme active site for substrate incorporation. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Todokoro T.,Research Institute Gekkeikan Sake Co. | Fukuda K.,Research Institute Gekkeikan Sake Co. | Matsumura K.,Research Institute Gekkeikan Sake Co. | Irie M.,Research Institute Gekkeikan Sake Co. | Hata Y.,Research Institute Gekkeikan Sake Co.
Journal of the Science of Food and Agriculture | Year: 2015

BACKGROUND: Deferriferrichrysin (Dfcy) is a siderophore found in foods fermented by Aspergillus oryzae and is a promising candidate for an antioxidant food additive because of its high binding constant toward iron. However, the Dfcy concentration is typically low in foods and cultures. RESULTS: We optimised culture conditions to improve Dfcy production to 2800mg L-1 from 22.5mg L-1 under typical conditions. Then, we evaluated the potential of Dfcy as a food additive by measuring its safety, stability, and antioxidant activity. Dfcy was sufficiently stable that over 90% remained after pasteurisation at 63°C for 30min at pH 3-11, or after sterilisation at 120°C for 4min at pH 4-6. Dfcy showed high antioxidant activity in an oil-in-water model, where inhibition of lipid oxidation was measured by peroxide value (PV) and thiobarbituric acid reactive substances (TBARS) assays. Dfcy decreased PV and TBARS by 83% and 75%, respectively. Antioxidant activity of Dfcy was equal to or higher than that of the synthetic chelator EDTA. CONCLUSION: Our study provides the first practical method for production of Dfcy. Dfcy can be a novel food-grade antioxidant and the first natural alternative to the synthesised iron chelator EDTA. © 2015 Society of Chemical Industry.

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