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…

Moscatel vine-shoot extracts as grapevine biostimulant to increase the varietal aroma of Airén wines

There is a growing interest in the exploitation of vine-shoots waste, since they are often left or burned. Sánchez-Gómez et al. [1] have shown that vines-shoots aqueous extracts have significant contents of bioactive compounds, among which several polyphenols and volatiles are highlighted. Recent studied had demonstrated that the chemical composition of vine-shoots is enhanced when vine-shoots are toasted
[2,3]. The application of vegetable products in the vineyards has led to significant changes towards a more “Sustainable Viticulture”. An innovative foliar application for Airén vine-shoot extracts have been carried out to the vineyard. It has been shown that they act as grape biostimulants, improving certain wine quality characteristics [4].

Enological evaluation of the attitude of the grapevine fumin to give varietal wines

Initiatives have been ongoing in recent years to safeguard biodiversity in the oenological sector via a process of enhancement of ancient varieties, under a pressure of a market strongly oriented towards production deriving from native vines of specific geographical zones. In that sense, Aosta Valley
(Italy) has raised the need to preserve and characterize its minority vine varieties which have the potentiality to give varietal wines. Fumin represents the 7% of the production of the region with 16 hectares of vineyards and 753 hectolitres of derived wine. Due to its large phenolic potential, strong astringency and deep colour, it has long been, and is still today, assembled or blended with other varieties as occurs, for example, for the Torrette.

Development of a new sustainable filtering media for wine and beer clarification and sterilisation

Different separation techniques are frequently used during vinification process. Nowadays, clarification and microbiological stabilization of wine or beer can be done using precoat filters or crossflow filters to remove yeast and bacteria. Kieselguhr powders are the most used filter aids for precoat filtration. Their crystalline structure and their pulverulent nature induce ecotoxicological risks when used. Moreover, regeneration and reuse of these filter aids is not efficient and the filtration waste requires cost effective retreatment.

Impact of varying ethanol and carbonation levels on the odor threshold of 1,1,6-trimethyl-1,2-dihydronaphtalene (petrol off-flavor) and role of berry size and Riesling clones

1,1,6-trimethyl-1,2-dihydronaphtelene (TDN) evokes the odor of “petrol” in wine, especially in the variety Riesling. Increasing UV-radiation due to climate change intensifies formation of carotenoids in the berry skins and an increase of TDN-precursors1. Exploring new viticultural and oenological strategies to limit TDN formation in the future requires precise knowledge of TDN thresholds in different matrices. Thresholds reported in the literature vary substantially between 2 µg/L up to 20 µg/L2,3,4 due to the use of different methods. As Riesling grapes are used for very different wine styles such as dry, sweet or sparkling wines, it is essential to study the impact of varying ethanol and carbonation levels.

Comparative proteomic analysis of wines made from Botrytis cinerea infected and healthy grapes reveal interesting parallels to the gushing phenomenon in sparkling wine

In addition to aroma compounds also protein composition strongly influences the quality of wines. Proteins of wine derive mainly from the plant Vitis vinifera and may be influenced by abiotic stress as well as fermentation conditions or fining. Additionally, fungal infections can affect the protein content as well by introducing fungal proteins or affecting grape protein composition. An infection of the vine with the plant pathogenic fungus Botrytis (B.) cinerea was shown to cause a degradation of proteins in the resulting wine. Moreover, it influences the foaming properties in sparkling wine.