Production and characterisation of AoSOX2 from Aspergillus oryzae, a novel flavin-dependent sulfhydryl oxidase with good pH and temperature stability
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Sulfhydryl oxidases have found application in the improvement of both dairy and baking products due to their ability to oxidise thiol groups in small molecules and cysteine residues in proteins. A genome mining study of the available fungal genomes had previously been performed by our group in order to identify novel sulfhydryl oxidases suitable for industrial applications and a representative enzyme was produced, AoSOX1 from Aspergillus oryzae (Faccio et al. BMC Biochem 11:31, 2010). As a result of the study, a second gene coding for a potentially secreted sulfhydryl oxidase, AoSOX2, was identified in the genome of A. oryzae. The protein AoSOX2 was heterologously expressed in Trichoderma reesei and characterised with regard to both biochemical properties as well as preliminary structural analysis. AoSOX2 showed activity on dithiothreitol and glutathione, and to a lesser extent on D/L-cysteine and beta-mercaptoethanol. AoSOX2 was a homodimeric flavin-dependent protein of approximately 78 kDa (monomer 42412 Da) and its secondary structure presents alpha-helical elements. A. oryzae AoSOX2 showed a significant stability to pH and temperature.
KeywordsSulfhydryl oxidase Secreted Fungal Flavoenzyme Production Characterization
The work was funded by the Marie Curie EU-project PRO-ENZ (MEST-CT-2005-020924) and by a personal grant to GF by the Finnish Cultural Foundation. We are thankful to Harry Boer and Evanthia Monogioudi for the assistance in CD spectroscopy and mass spectrometry. The technical assistance of Hanna Kuusinen is also acknowledged. The authors declare that they have no conflict of interest.
- Haarasilta S, Pillinene T, Vaisanen S, Tammersalo-Karsten I (1991) Enzyme product and method of improving the properties of dough and the quality of bread. European Patent EP0321811.Google Scholar
- Haarasilta S, Vaisanen S (1989) Method for improving flour dough. 88120669.2. European Patent 0321811A1.Google Scholar
- Hanes CS (1932) Studies on plant amylases: the effect of starch concentration upon the velocity of hydrolysis by the amylase of germinated barley. Biochem J 26:1406–1421Google Scholar
- Janolino VG, Swaisgood HE (1975) Isolation and characterization of sulfhydryl oxidase from bovine milk. J Biol Chem 250:2532–2538Google Scholar
- Kaufman SP, Fennema O (1987) Evaluation of sulfhydryl oxidase as a strengthening agent for wheat flour dough. Cereal Chem 64:172–176Google Scholar
- Kusakabe H, Kuninaka A, Yoshino H (1982) Purification and properties of a new enzyme, glutathione oxidase from Penicillium sp. K-6-5. Agric Biol Chem 46:2057–2067Google Scholar
- Kusakabe H, Midorikawa Y, Kuninaka A, Yoshino H (1983) Distribution of oxygen related enzymes in molds. Agric Biol Chem 47:1385–1387Google Scholar
- Macheroux P (1999) UV-visible spectroscopy as a tool to study flavoproteins. Meth Mol Biol 131:1–7Google Scholar
- Massey V, Müller F, Feldberg R, Schuman M, Sullivan PA, Howell LG, Mayhew SG, Matthews RG, Foust GP (1969) The reactivity of flavoproteins with sulfite. possible relevance to the problem of oxygen reactivity. J Biol Chem 244:3999–4006Google Scholar
- Neufeld HA, Green LF, Latterell FM, Weintraub RL (1958) Thioxidase, a new sulfhydryl-oxidizing enzyme from Piricularia oryzae and Polyporus versicolor. J Biol Chem 232:1093–1099Google Scholar
- Swaisgood HE (1977) Process of removing the cooked flavour from milk. United States Patent 4053644.Google Scholar