Doctoral Degrees (Institute for Wine Biotechnology)

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Browsing Doctoral Degrees (Institute for Wine Biotechnology) by Subject "Aspartic protease (MpAPr1)"

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    Investigating the impact of MpAPr1, an aspartic protease from the yeast Metschnikowia pulcherrima, on wine properties
    (Stellenbosch : Stellenbosch University, 2017-03) Theron, Louwrens Wiid; Divol, Benoit; Bely, Marina; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    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 actively sought 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 one such alternative. Recent literature reports the successful outcome of the addition of fungal aspartic proteases from Aspergillus and Botrytis. In this study, MpAPr1, an extracellular aspartic protease previously isolated and partially characterised from the yeast Metschnikowia pulcherrima, was cloned and expressed heterologously in Komagataella pastoris. Enzymatic properties of MpAPr1 were initially (Km, Vmax, K’i, optimal pH and temperature for protease activity, impact of minerals, sugars and ethanol on protease activity) characterised in a crude extract. After several attempts using different techniques, MpAPr1 was successfully purified via cation exchange chromatography. Its activity against haze-forming grape proteins was initially tested in a model solution under optimal environmental conditions (for MpAPr1 activity) and under those occurring during winemaking (pH 3.5 and 25°C). Thereafter, MpAPr1 activity was evaluated in grape must and throughout alcoholic fermentation. These experiments showed that MpAPr1 was able to degrade certain haze-forming proteins, especially chitinases, under optimal conditions and to a lesser extent under winemaking conditions. Prior denaturation of the target proteins by heat treatment was also not required. Moreover, MpAPr1 was able to degrade yeast proteins in a model solution under both conditions. Finally, the presence of MpAPr1, supplemented to grape must, resulted in the partial degradation of grape proteins throughout fermentation 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 on enhancing MpAPr1 activity to make it a viable alternative to bentonite. The study nevertheless provides further evidence that aspartic proteases could represent a potential alternative to bentonite for the wine industry and that non-Saccharomyces yeasts such as M. pulcherrima could have a beneficial impact on wine properties.