title: Cloning the putative gene of vinyl phenol reductase of Dekkera bruxellensis in Saccharomyces cerevisiae.
abstract: Vinylphenol reductase of Dekkera bruxellensis, the characteristic enzyme liable for "Brett" sensory modification of wine, has been recently recognized to belong to the short chain dehydrogenases/reductases family. Indeed, a preliminary biochemical characterisation has conferred to the purified protein a dual significance acting as superoxide dismutase and as a NADH-dependent reductase. The present study aimed for providing a certain identification of the enzyme by cloning the VPR gene in S. cerevisiae, a species not producing ethyl phenols. Transformed clones of S. cerevisiae resulted capable of expressing a biologically active form of the heterologous protein, proving its role in the conversion of 4-vinyl guaiacol to 4-ethyl guaiacol. A VPR specific protein activity of 9 ± 0.6 mU/mg was found in crude extracts of S. cerevisiae recombinant strain. This result was confirmed in activity trials carried out with the protein purified from transformant cells of S. cerevisiae by a his-tag purification approach; in particular, VPR-enriched fractions showed a specific activity of 1.83 ± 0.03 U/mg at pH 6.0. Furthermore, in agreement with literature, the purified protein behaves like a SOD, with a calculated specific activity of approximatively 3.41 U/mg. The comparative genetic analysis of the partial VPR gene sequences from 17 different D. bruxellesis strains suggested that the observed polymorphism (2.3%) and the allelic heterozygosity state of the gene do not justify the well described strain-dependent character in producing volatile phenols of this species. Actually, no correlation exists between genotype membership of the analysed strains and their capability to release off-flavours. This work adds valuable knowledge to the study of D. bruxellensis wine spoilage and prepare the ground for interesting future industrial applications.
authors.affiliation: Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, Italy.
authors.forename: Diego
authors.initials: D
authors.lastname: Romano
authors.affiliation: Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Valdetara
authors.affiliation: Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Zambelli
authors.affiliation: Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Galafassi
authors.affiliation: Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, Italy.
authors.forename: Valerio
authors.initials: V
authors.lastname: De Vitis
authors.affiliation: Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Molinari
authors.affiliation: Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, Italy.
authors.forename: Concetta
authors.initials: C
authors.lastname: Compagno
authors.affiliation: Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Foschino
authors.affiliation: Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, Italy. Electronic address: ileana.vigentini@unimi.it.
authors.forename: Ileana
authors.initials: I
authors.lastname: Vigentini
mesh_terms: D003001:Cloning, Molecular; D055167:Dekkera / Q000201:enzymology / Q000235:genetics*; D005285:Fermentation; D005516:Food Microbiology; D005838:Genotype; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010636:Phenols / Q000378:metabolism; D011110:Polymorphism, Genetic; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D014920:Wine / Q000032:analysis
chemical_list: D010636:Phenols; D011994:Recombinant Proteins; D010088:Oxidoreductases
keywords: B. bruxellensis; Genetic polymorphism; VPR enzyme; Volatile phenols; Wine