Applied Microbiology and Biotechnology

, Volume 90, Issue 3, pp 941–949 | Cite as

Production and characterisation of AoSOX2 from Aspergillus oryzae, a novel flavin-dependent sulfhydryl oxidase with good pH and temperature stability

  • Greta Faccio
  • Kristiina Kruus
  • Johanna Buchert
  • Markku Saloheimo
Biotechnologically Relevant Enzymes and Proteins


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.


Sulfhydryl 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.


  1. Alejandro S, Rodriguez PL, Belles JM, Yenush L, Garcia-Sanchez MJ, Fernandez JA, Serrano R (2007) An Arabidopsis quiescin-sulfhydryl oxidase regulates cation homeostasis at the root symplast–xylem interface. EMBO J 26:3203–3215CrossRefGoogle Scholar
  2. Bardwell J (1991) Identification of a protein required for disulfide bond formation in vivo. Cell 67:581–589. doi: 10.1016/0092-8674(91)90532-4 CrossRefGoogle Scholar
  3. Belton P (1999) Mini review: on the elasticity of wheat gluten. J Cereal Sci 29:103–107CrossRefGoogle Scholar
  4. Coppock DL, Thorpe C (2006) Multidomain flavin-dependent sulfhydryl oxidases. Antioxid Redox Signal 8:300–311CrossRefGoogle Scholar
  5. de la Motte RS, Wagner FW (1987) Aspergillus niger sulfhydryl oxidase. Biochemistry 26:7363–7371CrossRefGoogle Scholar
  6. Faccio G, Kruus K, Buchert J, Saloheimo M (2010) Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae. BMC Biochem 11:31. doi: 10.1186/1471-2091-11-31 CrossRefGoogle Scholar
  7. Farrell SR, Thorpe C (2005) Augmenter of liver regeneration: a flavin-dependent sulfhydryl oxidase with cytochrome C reductase activity. Biochemistry 44:1532–1541. doi: 10.1021/bi0479555 CrossRefGoogle Scholar
  8. Gerber J, Mühlenhoff U, Hofhaus G, Lill R, Lisowsky T (2001) Yeast ERV2p is the first microsomal FAD-linked sulfhydryl oxidase of the Erv1p/Alrp protein family. J Biol Chem 276:23486–23491CrossRefGoogle Scholar
  9. Ghanem M, Gadda G (2006) Effects of reversing the protein positive charge in the proximity of the flavin N(1) locus of choline oxidase. Biochemistry 45:3437–3447. doi: 10.1021/bi052514m CrossRefGoogle Scholar
  10. 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
  11. Haarasilta S, Vaisanen S (1989) Method for improving flour dough. 88120669.2. European Patent 0321811A1.Google Scholar
  12. 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
  13. Heckler EJ, Alon A, Fass D, Thorpe C (2008) Human quiescin-sulfhydryl oxidase, QSOX1: probing internal redox steps by mutagenesis. Biochemistry 47:4955–4963CrossRefGoogle Scholar
  14. Hoober KL, Joneja B, III White HB, Thorpe C (1996) A sulfhydryl oxidase from chicken egg white. J Biol Chem 271:30510–30516CrossRefGoogle Scholar
  15. Hoober KL (1999) Homology between egg white sulfhydryl oxidase and quiescin Q6 defines a new class of flavin-linked sulfhydryl oxidases. J Biol Chem 274:31759. doi: 10.1074/jbc.274.45.31759 CrossRefGoogle Scholar
  16. Hoober KL, Sheasley SL, Gilbert HF, Thorpe C (1999) Sulfhydryl oxidase from egg white. A facile catalyst for disulfide bond formation in proteins and peptides. J Biol Chem 274:22147–22150CrossRefGoogle Scholar
  17. Ito K, Inaba K (2008) The disulfide bond formation (Dsb) system. Curr Opin Struct Biol 18:450–458CrossRefGoogle Scholar
  18. Jaje J, Wolcott HN, Fadugba O, Cripps D, Yang AJ, Mather IH, Thorpe C (2007) A flavin-dependent sulfhydryl oxidase in bovine milk. Biochemistry 46:13031–13040CrossRefGoogle Scholar
  19. Janolino VG, Barnes CS, Swaisgood HE (1980) Dissociation and unfolding of sulfhydryl oxidase in solutions of guanidinium chloride. J Dairy Sci 63(12):1969–1974CrossRefGoogle Scholar
  20. Janolino VG, Swaisgood HE (1975) Isolation and characterization of sulfhydryl oxidase from bovine milk. J Biol Chem 250:2532–2538Google Scholar
  21. Kaufman SP, Fennema O (1987) Evaluation of sulfhydryl oxidase as a strengthening agent for wheat flour dough. Cereal Chem 64:172–176Google Scholar
  22. Kontkanen H, Westerholm-Parvinen A, Saloheimo M, Bailey M, Rättö M, Mattila M, Mohsina M, Kalkkinen N, Nakari-Setälä T, Buchert J (2009) Novel Coprinopsis cinerea polyesterase that hydrolyzes cutin and suberin. Appl Environ Microbiol 75:2148–2157CrossRefGoogle Scholar
  23. 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
  24. Kusakabe H, Midorikawa Y, Kuninaka A, Yoshino H (1983) Distribution of oxygen related enzymes in molds. Agric Biol Chem 47:1385–1387Google Scholar
  25. Lee J (2000) Erv1p from Saccharomyces cerevisiae is a FAD-linked sulfhydryl oxidase. FEBS Lett 477:62–66. doi: 10.1016/S0014-5793(00)01767-1 CrossRefGoogle Scholar
  26. Levitan A, Danon A, Lisowsky T (2004) Unique features of plant mitochondrial sulfhydryl oxidase. J Biol Chem 279:20002–20008CrossRefGoogle Scholar
  27. Lisowsky T (2001) Mammalian augmenter of liver regeneration protein is a sulfhydryl oxidase. Dig Liver Dis 33:173–180. doi: 10.1016/S1590-8658(01)80074-8 CrossRefGoogle Scholar
  28. Macheroux P (1999) UV-visible spectroscopy as a tool to study flavoproteins. Meth Mol Biol 131:1–7Google Scholar
  29. Martin JL (1995) Thioredoxin—a fold for all reasons. Structure 3:245–250CrossRefGoogle Scholar
  30. 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
  31. 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
  32. Ostrowski MC, Kistler WS (1980) Properties of a flavoprotein sulfhydryl oxidase from rat seminal vesicle secretion. Biochemistry 19:2639–2645CrossRefGoogle Scholar
  33. Penttila M, Nevalainen H, Ratto M, Salminen E, Knowles J (1987) A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene 61:155–164CrossRefGoogle Scholar
  34. Raje S, Glynn NM, Thorpe C (2002) A continuous fluorescence assay for sulfhydryl oxidase. Anal Biochem 307:266–272CrossRefGoogle Scholar
  35. Rosenfeld J, Capdevielle J, Guillemot JC, Ferrara P (1992) In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal Biochem 203:173–179CrossRefGoogle Scholar
  36. Shewry P (2000) Wheat glutenin subunits and dough elasticity: findings of the EUROWHEAT project. Trends Food Sci Technol 11:433–441CrossRefGoogle Scholar
  37. Shewry PR, Halford NG, Belton PS, Tatham AS (2002) The structure and properties of gluten: an elastic protein from wheat grain. Philos Trans R Soc Lond B Biol Sci 357:133–142. doi: 10.1098/rstb.2001.1024 CrossRefGoogle Scholar
  38. Swaisgood HE (1977) Process of removing the cooked flavour from milk. United States Patent 4053644.Google Scholar
  39. Tury A, Mairet-Coello G, Esnard-Feve A, Benayoun B, Risold PY, Griffond B, Fellmann D (2006) Cell-specific localization of the sulphydryl oxidase QSOX in rat peripheral tissues. Cell Tissue Res 323:91–103CrossRefGoogle Scholar
  40. Vala A, Sevier CS, Kaiser CA (2005) Structural determinants of substrate access to the disulfide oxidase Erv2p. J Mol Biol 354:952–966. doi: 10.1016/j.jmb.2005.09.076 CrossRefGoogle Scholar
  41. Vignaud C, Kaid N, Rakotozafy L, Davidou S, Nicolas J (2002) Partial purification and characterization of sulfhydryl oxidase from Aspergillus niger. J Food Sci 67:2016–2022CrossRefGoogle Scholar
  42. Wang C, Wesener SR, Zhang H, Cheng YQ (2009) An FAD-dependent pyridine nucleotide-disulfide oxidoreductase is involved in disulfide bond formation in FK228 anticancer depsipeptide. Chem Biol 16:585–593CrossRefGoogle Scholar
  43. Wu CK, Dailey TA, Dailey HA, Wang BC, Rose JP (2003) The crystal structure of augmenter of liver regeneration: a mammalian FAD-dependent sulfhydryl oxidase. Protein Sci 12:1109–1118CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Greta Faccio
    • 1
  • Kristiina Kruus
    • 1
  • Johanna Buchert
    • 1
  • Markku Saloheimo
    • 1
  1. 1.VTT Technical Research Centre of FinlandEspooFinland

Personalised recommendations