Evidence for an Extracellular Acid Proteolytic Activity Secreted by Living Cells ofSaccharomyces cerevisiaePlR1: Impact on Grape Proteins

Abstract

In this work, Saccharomyces cerevisiae PlR1, a strain isolated from Pinot noir grapes in the Champagne area, was shown to secrete an acid proteolytic activity against bovine serum albumin. This proteolytic activity was detectable in cell-free culture supernatants at the beginning of the exponential growth phase and increased with yeast growth. Using a zymography method, only one protease band with a molecular mass of 72 kDa was observed. This extracellular proteolytic activity was detected in the pH range from 2 to 4 with a maximal value at pH 2.5 and 38 °C and was completely inhibited by pepstatin A. The secretion of this protease did not need any protein inducer and seemed to be insensitive to nitrogen catabolic repression. S. cerevisiae PlR1 was also able to secrete this proteolytic activity during alcoholic fermentation, and it was found to be active against grape proteins, with a molecular mass around 25 kDa, at optimal conditions of 38 °C, pH 3.5.

Enzymatic hydrolysis of thermo-sensitive grape proteins by a yeast protease as revealed by a proteomic approach

“…Two proteins VVTL1 (gi|2213852) and a class IV endochitinase (gi|2306811) which belong, respectively, to the PR-5 and the PR-3 families, have already been identified in our previous work (Younes et al, 2011) and were sensitive to hydrolysis by the S. cerevisiae PlR1 proteolytic activity secreted during alcoholic fermentation. Two new proteins: a putative thaumatin-like (gi|7406716), which belongs to the PR-5 family and a hypothetical protein (gi|225426795) are also characterized.…”

“…Yeast strain, culture conditions and supernatant preparation S. cerevisiae PlR1 has been isolated following spontaneous fermentation of a Pinot noir grape juice. Culture conditions and supernatant preparation were as previously described (Younes et al, 2011). Proline was used as the only nitrogen source for yeast growth.…”

“…Contrary to non-Saccharomyces yeast strains, fermentative yeast of the Food Research International xxx (2013) xxx-xxx genus Saccharomyces do not secrete any acidic proteolytic activity (Ogrydziak, 1993). However, Saccharomyces cerevisiae PlR1, a wild yeast strain isolated from Pinot noir grapes, was recently shown to secrete an acidic proteolytic activity whose optimal pH and temperature conditions were 2.5 and 38°C, respectively (Younes et al, 2011).…”

Enzymes for Wine Fermentation: Current and Perspective Applications

“…Most Saccharomyces cerevisiae strains show no extracellular protease activity on diagnostic agar media [16][17][18][19]69]. However, a 72 kDa extracellular pepsin-like aspartic protease was characterized from a PIR 1 strain [70,71]. The enzyme was active during grape juice fermentations, although it did not affect turbidity-inducing proteins, unless the wine was incubated at 38 • C for extended time.…”

Degradation of white wine haze proteins by Aspergillopepsin I and II during juice flash pasteurization

“…Several authors have investigated the effects of microbial proteases such as those from Aspergillus niger (Bakalinsky & Boulton, 1985), Saccharomyces cerevisiae (Dizy & Bisson, 2000;Feuillat, Brillant, & Rochard, 1980;Lagace & Bisson, 1990;Younes et al, 2011), and Botrytis cinerea (Cilindre, Castro, Clément, Jeandet, & Marchal, 2007;Girbau et al, 2004;Marchal et al, 1998). However, in each study, the enzymes were not able to effectively degrade grape PR proteins because of their high proteolysis resistance and because winemaking temperature conditions are unfavourable for enzyme activity (Waters, Peng, Pocock, & Williams, 1995;Waters et al, 1992).…”