Browsing by Author "Theron, Louwrens Wiid"
Now showing 1 - 2 of 2
Results Per Page
Sort Options
- ItemExpression and purification of recombinant extracellular proteases originating from non-Saccharomyces yeasts(Stellenbosch : Stellenbosch University, 2013-12) Theron, Louwrens Wiid; Divol, Benoit; Zietsman, Anscha; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: During wine fermentation, yeasts release extracellular enzymes that significantly impact wine properties. While the extracellular proteins of Saccharomyces cerevisiae have been characterised, those of non-Saccharomyces yeasts remain largely unknown. Most of these enzymes break down sugar polymers or catalyse the liberation of glycosidically-bound molecules. Another category of enzymes of oenological interest is represented by acid proteases that are able to prevent or reduce protein haze, as reported in literature, while simultaneously increasing the assimilable nitrogen content of wine. The liberation of amino acids from peptides and proteins that serve as aroma precursors may also have an indirect effect on wine aroma. In a recent study performed at the Institute for Wine Biotechnology (IWBT), the sequences of two aspartic proteases were retrieved from non-Saccharomyces yeast species isolated from South African wines. The genes, MpAPr1 and CaAPr1, were isolated from two non-Saccharomyces species, Metschnikowia pulcherrima IWBT Y1123 and Candida apicola IWBT Y1384, respectively. However, no further characterization was undertaken. This study aimed to clone these two genes into a recombinant bacterial host for expression and purify the corresponding enzymes as a first step toward characterizing their kinetic properties. Considering that some non-Saccharomyces species have been shown to produce more than one acid protease, an additional aim was to identify novel acid proteases within M. pulcherrima IWBT Y1123. Cloning of the genes and transformation of the expression vectors into E. coli were achieved. Optimal conditions for induced expression were established following extensive optimization. Furthermore, while native extraction of the recombinant proteins was unsuccessful, denaturing conditions allowed their recovery, suggesting that the recombinant proteins are encapsulated into inclusion bodies. Recombinant MpAPr1 was purified by using a nickel based column system and mass fingerprinting of the purified enzyme (MpAPr1) confirmed its identity. Purification was followed by refolding experiments, but yielded poor recovery of active enzymes. Unfortunately, recombinant expression of CaAPr1 could not be observed for reasons yet to be elucidated that may include the large sequence dissimilarities between CaAPr1 and MpAPr1. Finally, Southern blot analysis on the genomes of M. pulcherrima IWBT Y1123 and C. apicola IWBT Y1384 revealed that both possess at least one additional protease other than those previously described. Further analysis of the extracellular proteome of M. pulcherrima IWBT Y1123 also confirmed the presence of at least one enzyme able to hydrolyze BSA at a low pH. Unfortunately, mass fingerprinting performed on the entire extracellular proteome and on small groups of proteins thereof did not allow the identification of these enzymes.
- ItemInvestigating 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.