Partial vinylphenol reductase purification and characterization from Brettanomyces bruxellensis.
Brettanomyces is the major microbial cause for wine spoilage worldwide and causes significant economic losses. The reasons are the production of ethylphenols that lead to an unpleasant taint described as 'phenolic odour'. Despite its economic importance, Brettanomyces has remained poorly studied at the metabolic level. The origin of the ethylphenol results from the conversion of vinylphenols in ethylphenol by Brettanomyces hydroxycinnamate decarboxylase. However, no information is available on the vinylphenol reductase responsible for the conversion of vinylphenols in ethylphenols. In this study, a vinylphenol reductase was partially purified from Brettanomyces bruxellensis that was active towards 4-vinylguaiacol and 4-vinylphenol only among the substrates tested. First, a vinylphenol reductase activity assay was designed that allowed us to show that the enzyme was NADH dependent. The vinylphenol reductase was purified 152-fold with a recovery yield of 1.77%. The apparent K(m) and V(max) values for the hydrolysis of 4-vinylguaiacol were, respectively, 0.14 mM and 1900 U mg(-1). The optimal pH and temperature for vinylphenol reductase were pH 5-6 and 30 degrees C, respectively. The molecular weight of the enzyme was 26 kDa. Trypsic digest of the protein was performed and the peptides were sequenced, which allowed us to identify in Brettanomyces genome an ORF coding for a 210 amino acid protein.
Tchobanov
Iavor
I
Institut Universitaire de Vigne et du Vin Jules Guyot, Université de Bourgogne, Dijon Cedex, France.
Gal
Laurent
L
Guilloux-Benatier
Michèle
M
Remize
Fabienne
F
Nardi
Tiziana
T
Guzzo
Jean
J
Serpaggi
Virginie
V
Alexandre
Hervé
H
D000595:Amino Acid Sequence; D002849:Chromatography, Gas; D004591:Electrophoresis, Polyacrylamide Gel; D005516:Food Microbiology; D005656:Fungal Proteins / Q000302:isolation & purification* / Q000378:metabolism; D006139:Guaiacol / Q000031:analogs & derivatives / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D016366:Open Reading Frames; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D010636:Phenols / Q000378:metabolism*; D004718:Saccharomycetales / Q000201:enzymology*; D013696:Temperature; D014920:Wine / Q000382:microbiology
D005656:Fungal Proteins; D010636:Phenols; D009243:NAD; D006139:Guaiacol; C014245:4-vinylguaiacol; C042291:4-ethylphenol; C084991:4-ethylguaiacol; D010088:Oxidoreductases; C030626:4-vinylphenol