Macrowine 2021
IVES 9 IVES Conference Series 9 Proteomic and activity characterization of exocellular laccases from three Botrytis cinerea strains

Proteomic and activity characterization of exocellular laccases from three Botrytis cinerea strains

Abstract

Botrytis cinerea is a fungus that causes common infection in grapes and other fruits. In winemaking, its presence can be both considered desirable in the case of noble rot infection or undesirable when grey rot is developed. This fungus produces an extracellular enzyme known as laccase which is able to cause oxidation of phenolic compounds present in must and wine, causing most of the times a decrease in its quality and problems during the winemaking process [1]. Material and methods: Three B. cinerea strains (B0510, VA612 and RM344) were selected and grown in a liquid medium adapted from one previously described [2]. The enzyme was isolated by tangential ultrafiltration of the culture medium using a QuixStand system equipped with a 30 KDa filtration membrane. The purity of the isolated enzymes was checked using SDS-PAGE. The characteristics (molecular weight, % of glycosylation, specific activity, activity in function of pH of the enzyme isolated from each strain were studied with ABTS as a substrate. Results: The enzymes isolated from the three strains showed the same molecular weight, 97 KDa, in good agreement with the molecular weight previously determined for B. cinerea laccase using SDS-PAGE [2]. The percentage of glycosylation was high, being estimated in 70% on weight, also similar to that described by other authors [3]. Despite similar physical characteristics of the enzymes obtained from different strains, their activity were quite different. The enzymes isolated from B0510 and VA612 strains showed similar specific activity for ABTS oxidation, being 0.3 and 0.21 mM for their Km and their Vmax were 1.28 and 1.45 mM/min per milligram of enzyme respectively. The activity for RM344 enzyme was found much lower, with values of 0.78 mM for Km and 0.13 mM/min per milligram of enzyme for Vmax. The enzyme isolated from the B0510 strain presented its highest activity at pH 2.9 while VA612 and RM344 enzymes showed the maximum activity at pH 3.3. All these values were quite lower compared to previously measured by other authors [4-6]. Those differences in the enzyme activity may be related with differences in the active center of the enzyme and could have important consequences for the winemaking process depending on the strains of the B. cinerea strain involved in the infection of grapes.

[1]P. Ribéreau-Gayon, Y. Glories, A. Maujean, D. Dubourdieu, Handbook of Enology, Volume 2: The chemistry of Wine Stabilization and Treatments, John Wiley & Sons, Ltd, 2000. [2]D. Slomczynski, J.P. Nakas, S.W. Tanenbaum, Applied and Environmental Microbiology, 61 (1995) 907. [3]C. THURSTON, Microbiology-Sgm, 140 (1994) 19. [4]I. MARBACH, E. HAREL, A. MAYER, Phytochemistry, 23 (1984) 2713. [5]I. MARBACH, E. HAREL, A. MAYER, Phytochemistry, 22 (1983) 1535. [6]M. Dubernet, P. Ribereau-Gayon, H.R. Lerner, E. Harel, A.M. Mayer, Phytochemistry, 16 (1977) 191.

Publication date: May 17, 2024

Issue: Macrowine 2016

Type: Poster

Authors

Cédric Saucier*, Anne-Sophie Walker, Christiane Auclair, François Garcia, Francois-Xavier Sauvage, Jullien Drone, Natalia Quijada-Morin, Patrick Chemardin

*Université de Montpellier

Contact the author

Tags

IVES Conference Series | Macrowine | Macrowine 2016

Citation

Related articles…

Evaluation of colloidal stability in white and rosé wines investing Dynamic Light Scattering technology

Proteins constitute one of the three main components of grape juice and white wine, phenolic compounds and polysaccharides being the others. A specific group of the total grape-derived proteins resists degradation or adsorption during the winemaking process and remains in finished wine if not removed by the commonplace commercial practice of bentonite fining. While bentonite is effective in removing the problematic proteins, it is claimed to adversely affect the quality of the treated wine under certain conditions, through the removal of colour, flavor and texture compounds. A number of studies have indicated that different protein fractions require distinct bentonite concentrations for protein removal and consequent heat stabilization.

Effect of intra‐vineyard ripeness variation on the efficiency of commercial enzymes on berry cell wall deconstruction under winemaking conditions

Intra-vineyard variation grape berry ripening occurs within bunches, between bunches on the same vine and between vines. Although it is assumed that such variation also occurs at the grape berry cell wall level, no study to data has investigated in any depth. Here we have used a intra-vineyard panel design to investigate pooled bunches from six vines (per panel) in the context of a winemaking scenario. The dissected vineyard was harvested by separate panels, where each panel was then subjected to a standard winemaking procedure with or without the addition of three different enzyme preparations for maceration.

Partial dealcoholisation of red wine by reverse osmosis-evaporative perstraction: impact on wine composition

Around the world, the alcohol content of wine has been steadily increasing; partly as a consequence of climate change, but also due to improvements in viticultural management practices and winemaking techniques [1,2]. Concurrently, market demand for wines with lower alcohol levels has increased as consumers seek to reduce alcohol intake for social and/or health reasons [3]. As such, there is increasing demand for both innovative methods that allow winemakers to produce ‘reduced alcohol wines’ (RAW) and a better understanding of the impact of such methods on the composition of RAW. This study therefore aimed to investigate compositional changes in two red wines resulting from partial alcohol removal following treatment by one such method, involving a combination of reverse osmosis and evaporative perstraction (RO-EP).

Influence of wood chips addition during alcoholic fermentation on wine phenolic composition

This study investigates the effect of wood chips addition during the alcoholic fermentation on the phenolic
composition of the produced wines. A series of wood chips, originating from American, French, Slavonia
oak and Acacia were added at the beginning of wine alcoholic fermentation. Besides, a mixture consisting
of 50% French and 50% Americal oak chips were added during the experimentation. The wine samples
were analyzed one month after the end of malolactic fermentation, examining various chemical
parameters such as total anthocyanins, total phenolic content, tannins combined with protein (BSA) and
ellagitannin content.

Impact of elemental sulfur (S0) residues in Sauvignon blanc juice on the formation of the varietal thiols 3-mercapto hexanol and 3-mercaptohexyl acetate

Elemental sulfur is a fungicide used by grape growers to control the development of powdery mildew, caused by the fungus Erysiphe necator. This compound is effective, cheap and has a low toxicity with no withholding period recommended. However, high levels of S0 residues in the harvested grapes can lead to the formation of reductive sulfur compounds that can impart taints and faults to the wine. Hydrogen sulphide (H2S) is a very volatile and unpleasant sulfur compound which formation is connected to high residues of S0 in juice (10 – 100 mg/L).