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…

Oenological features of Sangiovese wine from vinification of whole grape berries

The present study was performed in a traditional winery located in the viticultural area of Brunello di Montalcino, Siena, Italy, in the vintage 2015. Actually, in this winery Sangiovese grape musts are fermented in large oak barrels by a single strain of Saccharomyces cerevisiae previously isolated in the same winery. Pumping over operations are carried out once or twice a day until the end of alcoholic fermentations. The aim of this work was to investigate on the oenological properties of Sangiovese wine produced with the traditional winemaking process adopted by the winery under study obtained from the fermentation of whole berries compared to that from crushed grape must. In particular, two lots of 65q of Sangiovese grapes from the same 3ha vineyard were vinified in 150hL oak barrels.

Ethyl esters interact with the major wine Thaumatin Like Protein VVTL1

The interactions among aromatic compounds and proteins is an important issue for the quality of foods and beverages. In wine, the loss of flavor after vinification is associated to bentonite treatment and this effect can be the result of the removal of aroma compounds which are bound wine proteins. This phenomenon was recently demonstrated for long chain fatty acids and their ethyl esters (1). Since these latter compounds are spectroscopically silent, their association with proteins is not easy to measure.

HEAT BERRY : Sensitivity of berries ripening to higher temperature and impact on phenolic compounds in wine

The grapevine is an important economical crop that is very sensitive to climate changes and microclimate. The observations made during the last decades at a vineyard scale all concur to show the impact of climate change on vine physiology, resulting in accelerated phenology and earlier harvest (Jones and Davis 2000). It is well-known that berry content is affected by the ambient temperature. While the first experiences were primarily conducted on the impact of temperature on anthocyanin accumulation in the grape, few studies have focused on others component of phenolic metabolism, such as tannins.

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.

Grape byproducts as source of resveratrol oligomers for the development of antifungal extracts

Grape canes are a non-recycled byproduct of wine industry (1-5 tons per hectare per year) containing valuable phytochemicals of medicine and agronomical interest. Resveratrol and wine polyphenols are known to exert a plethora of health-promoting effects including antioxidant capacity, cardioprotection, anticancer activity, anti-inflammatory effects, and estrogenic/antiestrogenic properties (Guerrero et al. 2009). Additionally, resveratrol is a major phytoalexin produced by plants in response to various stresses and promotes disease resistance (Chang et al. 2011). Our project aims to develop polyphenol-rich grape cane extracts to fight phytopathogenic or clinically relevant fungi. We initiate the project with the development of analytical methods to analyze resveratrol mono- and oligomers (dimers, trimers and tetramers) from grape canes and we evaluate their potential activity against clinically relevant opportunistic fungal pathogens (Houillé et al. 2014).