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…

Analysis of peptide fraction from white wines

Among nitrogen compounds included in white wines, the peptide fraction is certainly the least studied, however this fraction is quantitatively the most important (Feuillat, 1974). Existing studies concern the fraction below 1 kDa and only for white and sparkling wines (Bartolomé et al, 1997, Desportes et al 2000). In this report, we have developed methods to isolate peptides from reference white wines. Then, we have applied this methodology with bitter wine to answer a research question: is there a relation between peptides and the bitterness of white wine as for some cheese for example (Furtado, 1984)?

Comparison of various storage conditions to preserve polyphenols in red-grape pomace

Red grape pomace, a waste from wine production, can be valorised by extracting polyphenols, high-added value compounds used in cosmetics or oenology. For use at an industrial level, using green extraction techniques, pomace need to be stored before being processed. The aim of this study is to test various storage conditions in order to maintain high level of polyphenols over 180 days, while keeping storage cost economically interesting. In a first step, different storage conditions (ambient temperature or cooled (4°C) temperature, anaerobic (saturation with N2) or aerobic conditions, and addition of sulphur dioxide (SO2)) were compared on small samples (1 kg) packed in plastic pockets. The quality of storage was assessed by following the optical density of the pomace extract at 280 nm (DO 280 expressed as mg/l eq gallic acid), which is an indication of the amount of remaining extractable polyphenols.

Maturation of Agiorgitiko (Vitis vinifera) red wine on its wine lees: Impact on its phenolic composition

Maturation of wine on lees (often referred as sur lie) is a common practice applied by many winemakers around the world. In the past this method was applied mainly on white and/or sparkling wine production but recently also to red wine production. In our experiment, we matured red wine on wine lees of two origins: a) Light wine lees, collected after the completion of the alcoholic fermentation, b) Heavy lees, collected after the completion of the malolactic fermentation. The lees were free of off-odors and were added in the red wine in percentage 3% and 8%, simulating common winemaking addition. The maturation lasted in total six months and samples were collected for analysis after one, three and six months. During storage the lees were stirred.

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.