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Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae

Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae,10.118

Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae   (Citations: 3)
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BACKGROUND: Sulfhydryl oxidases are flavin-dependent enzymes that catalyse the formation of de novo disulfide bonds from free thiol groups, with the reduction of molecular oxygen to hydrogen peroxide. Sulfhydryl oxidases have been investigated in the food industry to remove the burnt flavour of ultraheat-treated milk and are currently studied as potential crosslinking enzymes, aiming at strengthening wheat dough and improving the overall bread quality. RESULTS: In the present study, potential sulfhydryl oxidases were identified in the publicly available fungal genome sequences and their sequence characteristics were studied. A representative sulfhydryl oxidase from Aspergillus oryzae, AoSOX1, was expressed in the fungus Trichoderma reesei. AoSOX1 was produced in relatively good yields and was purified and biochemically characterised. The enzyme catalysed the oxidation of thiol-containing compounds like glutathione, D/L-cysteine, beta-mercaptoethanol and DTT. The enzyme had a melting temperature of 57°C, a pH optimum of 7.5 and its enzymatic activity was completely inhibited in the presence of 1 mM ZnSO4. CONCLUSIONS: Eighteen potentially secreted sulfhydryl oxidases were detected in the publicly available fungal genomes analysed and a novel proline-tryptophan dipeptide in the characteristic motif CXXC, where X is any amino acid, was found. A representative protein, AoSOX1 from A. oryzae, was produced in T. reesei in an active form and had the characteristics of sulfhydryl oxidases. Further testing of the activity on thiol groups within larger peptides and on protein level will be needed to assess the application potential of this enzyme.
Journal: BMC Biochemistry - BMC BIOCHEM , vol. 11, no. 1, pp. 31-11, 2010
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    • ... et al. 2007; Farrell and Thorpe 2005; Fass 2008; Gross et al. 2002; Levitan et al. 2004; Rodriguez et al. 2006; Wang et al. 2007; Wu et al. 2003), the endoplasmic reticulum oxidase (Ero) family (Frand and Kaiser 1999; Gross et al. 2004; Hiniker and Bardwell 2004; Vitu et al. 2010), the quiescinsulfhydryl oxidase (QSOX) family (Coppock and Thorpe 2006; Jaje et al. 2007) and secreted fungal SOXs (de la Motte and Wagner 1987; Faccio et al. 2010, ...
    • ...Representative proteins considered are yeast Erv1 and Erv2 (identifier P27882 and Q12284, respectively; Ang and Lu 2009; Gerber et al. 2001; Gross et al. 2002), bovine QSOX (AAI49741.1; Jaje et al. 2007), and Ero1 (Q03103.1; Gross et al. 2004; Tavender and Bulleid 2010) and fungal AoSOX1 (Q2UA33; Faccio et al. 2010)...
    • ...L/D-cysteine Faccio et al. (2010) Aspergillus...
    • ...SOXs have been the subject of a genome mining study that showed the presence of candidate enzymes in many Aspergilli and other fungal species such as Magnaporthe grisea and Neosartorya fischeri (Faccio et al. 2010)...
    • ...(Tokyo, Japan). Recently, two novel SOXs from Aspergillus oryzae, AoSOX1 and AoSOX2, have been heterologously produced in Trichoderma reesei, and production levels of 70 and 180 mg/l, respectively, have been registered in shake flask cultivations (Faccio et al. 2010, 2011)...

    Greta Faccioet al. Sulfhydryl oxidases: sources, properties, production and applications

    • ...This same study that successfully lead to the production and biochemical characterisation of the secreted sulfhydryl oxidase AoSOX1 from Aspergillus oryzae (Faccio et al. 2010), identified in the genome of the fungus A. oryzae a second predicted protein, AoSOX2, with sequence features typical of sulfhydryl oxidases...
    • ...Sulfhydryl oxidase activity was also assayed with a peroxidasehomovanillic acid coupled assay modified from Raje et al. (2002) as described by Faccio et al. (2010)...
    • ...Fractions obtained from the purification steps were assayed for activity on glutathione with Ellman’s reagent (5,5′-dithiobis-(2-nitrobenzoic acid)) as reported (Faccio et al. 2010)...
    • ...Activity was measured with the peroxidase-homovanillic acid coupled assay (Faccio et al. 2010) in presence of different substrate concentrations and the kinetic parameters Km and Vmax were calculated by linear regression with the Hanes plot (Hanes 1932) using the graphing software Origin 7.5 SRO (OriginLab Corporation, Northampton, MA, USA)...
    • ...A genome mining study for secreted fungal sulfhydryl oxidases was previously conducted and yet lead to the identification of the secreted enzyme AoSOX1 from A. oryzae (Faccio et al. 2010)...
    • ...AoSOX1 from A. oryzae (50.6% identity; Faccio et al. 2010)...
    • ...The work presented here is the continuation of a previous genome mining study concerning fungal secreted sulfhydryl oxidases that lead yet to the production and biochemical characterisation of the sulfhydryl oxidase AoSOX1 from A. oryzae, recently characterised by our group (Faccio et al. 2010)...
    • ...The enzyme AoSOX2 was more resistant to chemical denaturation than the previously characterised AoSOX1 from A. oryzae (Faccio et al. 2010) and QSOX from milk (Janolino et al. 1980)...
    • ...AoSOX2 started to unfold at a concentration of guanidinium chloride (4.5 M) higher than the one reported for AoSOX1 (2 M) and QSOX (2 M; Faccio et al. 2010; Janolino et al. 1980)...
    • ...In addition, AoSOX2 showed a higher temperature stability than AoSOX1 since retained activity after 1 h of incubation at 60 °C. Moreover, the melting temperature of AoSOX2 around 75 °C was significantly higher than the one reported for AoSOX1 from A. oryzae (55 °C; Faccio et al. 2010) and the homodimeric enzyme Erv2 from yeast (59.2 °C; Vala et al. 2005)...
    • ...The physiological role of secreted sulfhydryl oxidases has not been clarified yet although few enzymes have been reported and, from A. oryzae in particular, two enzymes AoSOX1 (Faccio et al. 2010) and AoSOX2 have been characterised...

    Greta Faccioet al. Production and characterisation of AoSOX2 from Aspergillus oryzae , a ...

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