Investigating the impact of MpAPr1, an aspartic protease from the yeast Metschnikowia pulcherrima, on wine properties

[摘要] ENGLISH ABSTRACT: Protein removal is a key step during the production of white wine in order to avoid the possible appearance of a harmless but unsightly haze. Alternatives to the use of bentonite are activelysought because of technological, organoleptic and sustainable issues associated with its use.Acid proteases that are able to break down proteins under winemaking conditions could be onesuch alternative. Recent literature reports the successful outcome of the addition of fungalaspartic proteases from Aspergillus and Botrytis. In this study, MpAPr1, an extracellular asparticprotease previously isolated and partially characterised from the yeast Metschnikowiapulcherrima, was cloned and expressed heterologously in Komagataella pastoris. Enzymaticproperties of MpAPr1 were initially (Km, Vmax, K'i, optimal pH and temperature for proteaseactivity, impact of minerals, sugars and ethanol on protease activity) characterised in a crudeextract. After several attempts using different techniques, MpAPr1 was successfully purified viacation exchange chromatography. Its activity against haze-forming grape proteins was initiallytested in a model solution under optimal environmental conditions (for MpAPr1 activity) andunder those occurring during winemaking (pH 3.5 and 25°C). Thereafter, MpAPr1 activity wasevaluated in grape must and throughout alcoholic fermentation. These experiments showed thatMpAPr1 was able to degrade certain haze-forming proteins, especially chitinases, under optimalconditions and to a lesser extent under winemaking conditions. Prior denaturation of the targetproteins by heat treatment was also not required. Moreover, MpAPr1 was able to degrade yeastproteins in a model solution under both conditions. Finally, the presence of MpAPr1,supplemented to grape must, resulted in the partial degradation of grape proteins throughoutfermentation and ultimately in a slight difference in the wine's volatile compound composition.Winemaking conditions limited its impact and it is thus proposed that future work focus onenhancing MpAPr1 activity to make it a viable alternative to bentonite. The study neverthelessprovides further evidence that aspartic proteases could represent a potential alternative tobentonite for the wine industry and that non-Saccharomyces yeasts such as M. pulcherrimacould have a beneficial impact on wine properties.

[发布机构] Stellenbosch University