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Investigating the Impact of Mpapr1, an Aspartic Protease from the Yeast Metschnikowia Pulcherrima, on Wine Properties

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Investigating the Impact of Mpapr1, an Aspartic Protease from the Yeast Metschnikowia Pulcherrima, on Wine Properties THÈSE EN COTUTELLE PRÉSENTÉE POUR OBTENIR LE GRADE DE DOCTEUR DE L’UNIVERSITÉ DE BORDEAUX ET DE L’UNIVERSITÉ DE STELLENBOSCH ÉCOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTÉ SPÉCIALITÉ ŒNOLOGIE FACULTY OF AGRISCIENCES Par Louwrens THERON ETUDE DE L’IMPACT DE MPAPR1, UNE PROTEASE ASPARTIQUE DE LA LEVURE METSCHNIKOWIA PULCHERRIMA, SUR LES PROPRIETES DU VIN Sous la direction de Benoit DIVOL et de Marina BELY Soutenue le 27 janvier 2017 Membres du jury: Mme. LE HENAFF-LE MARREC Claire, Professeur à l’université de Bordeaux Président M. MARANGON Matteo, Chargé de recherche à l’université de Padoue Rapporteur Mme. CAMARASA Carole, Chargée de recherche à l’INRA de Montpellier Rapporteur M. BAUER Florian, Professeur à l’université de Stellenbosch Examinateur Titre : Etude de l’impact de MpAPr1, une protéase aspartique de la levure Metschnikowia pulcherrima, sur les propriétés du vin Résumé : L'élimination des protéines est une étape clé lors de la production du vin blanc afin d'éviter l'apparition éventuelle d'un voile inoffensif mais inesthétique. Des solutions de rechange à l'utilisation de la bentonite sont activement recherchées en raison des problèmes technologiques, organoleptiques et de durabilité associés à son utilisation. Dans cette étude, MpAPr1, une protéase aspartique extracellulaire préalablement isolée et partiellement caractérisée à partir de la levure Metschnikowia pulcherrima, a été clonée et exprimée de manière hétérologue dans la levure Komagataella pastoris. Les propriétés enzymatiques de MpAPr1 ont été initialement caractérisées dans un extrait brut. Après plusieurs essais faisant appel à différentes techniques, MpAPr1 a été purifié avec succès par chromatographie échangeusede cations. Son activité contre les protéines de raisin a été initialement testée dans une solution modèle dans des conditions environnementales optimales pour l'activité de MpAPr1 puis dans celles qui règnent lors de la vinification. Ensuite, l'activité de MpAPr1 a été évaluée dans du moût de raisin et au cours de la fermentation alcoolique. La présence de MpAPr1, supplémenté au moût de raisin, a entraîné une dégradation partielle des protéines de raisin tout au long de la fermentation et une légère différence dans la composition en composés volatils du vin. L'étude a confirmé que les protéases aspartiques pourraient représenter une alternative à la bentonite pour l'industrie du vin et que les levures non-Saccharomyces telles que M. pulcherrima pourraient avoir un impact bénéfique sur les propriétés du vin. Mots clés : Metschnikowia pulcherrima, protéase aspartique, caractérisation enzymatique, casse protéique, qualité organoleptique du vin Title : Investigating the impact of MpAPr1, an aspartic protease from the yeast Metschnikowia pulcherrima, on wine properties 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 sustainability issues associated with its use. 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 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. Thereafter, MpAPr1 activity was evaluated in grape must and throughout alcoholic fermentation. 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 composition in volatile compounds. The study 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. Keywords : Metschnikowia pulcherrima, aspartic protease, enzyme characterisation, protein haze, wine organoleptic quality Unité de recherche University of Stellenbosch, IWBT, Faculty of Agrisciences. 7602 Matieland Private Bag X1 Universite de Bordeaux, ISVV, EA 4577, Unite de Recherche Œnologie. 33140 Villenave d’Ornon Declaration By submitting this dissertation electronically, I declare that the entirety of the work contained therein is my own, original work, that I am the sole author thereof (save to the extent explicitly otherwise stated) that reproduction and publication thereof by Stellenbosch University will not infringe any third party rights and that I have not previously in its entirety or in part submitted it for obtaining any qualification. Date: 17 February 2017 Copyright © 2017 Stellenbosch University All rights reserved ii Summary 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. iii Opsomming Proteïen verwydering is 'n belangrike stap tydens die vervaardiging van witwyn en vermy die voorkoms van 'n onooglike maar skadelose wasigheid. Alternatiewe vir die gebruik van bentoniet is aktief begeerd as gevolg van tegnologiese, organoleptiese en volhoubare kwessies wat verband hou met die gebruik daarvan. Suur proteases wat die afbraak van proteïene kan fasiliteer onder wynmaak toestande kan dus as sulke alternatiewe dien. Onlangse literatuur beskryf die suksesvolle gebruik van swam-afkomstige aspartiensuur proteases vanaf Aspergillus en Botrytis. In hierdie studie was MpAPr1, ‘n ekstrasellulêre aspertiensuur protease voorheen geïsoleer en gedeeltelik gekenmerk vanaf die gis Metschnikowia pulcherrima, gekloneer en uitgedruk in Komagataella pastoris. Ensiem kenmerke (Km, Vmax, K’i, optimale pH and temperature vir protease aktiwiteit, impak van minerale, suiker en etanol) was aanvanklik bepaal vanaf ‘n ru-ekstrak. Na verskeie pogings deur gebruik te maak van verskeie tegnieke, is MpAPr1 suksesvol gesuiwer via katioonuitruilings chromatografie. Ensiem aktiwiteit teen waas-vormende druif proteïene was aanvanklik getoets in ‘n model oplossing onder optimale omgewings toestande (vir MpAPr1 aktiwiteit) en dié wat gedurende wynmaak voorkom (pH 3.5 en 25 °C). Daarna was MpAPr1 aktiwiteit geëvalueer in druiwemos tydens alkoholiese fermentasie. Eksperimente het getoon dat MpAPr1 verskeie waas-vormende proteïene, veral chitinases, afbreek onder optimale omstandighede en in 'n mindere mate onder wynmaak toestande. Voorafgaande denaturasie van die teiken proteïene deur hitte behandeling was nie benodig nie. Verdermeer was MpAPr1 instaat om gis proteïene af te breek in 'n model oplossing onder beide toestande. Ten slotte, die teenwoordigheid van MpAPr1 in druiwesap het gelei tot die gedeeltelike afbraak van proteïene tydens fermentasie asook tot 'n effense verskil in die uiteindelike vlugtige verbinding samestelling. Wynmaak toestande het gelei to beperkte ensiem impak en dus word dit voorgestel dat toekomstige werk fokus op die verbetering van MpAPr1 aktiwiteit
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