Investigating grape berry cell wall deconstruction by hydrolytic enzymes

Zietsman, (Anscha) Johanna Jacoba (2015-04)

Thesis (PhD)--Stellenbosch University, 2015.

Thesis

ENGLISH ABSTRACT: Maceration enzymes for the wine industry are preparations containing mainly pectinases, cellulases and hemicellulases, used during wine making to degrade the berry cell walls and release polyphenolic and aroma molecules to increase wine quality. These types of enzymes are also used for the harvesting of revenue-generating molecules from pomace (skins, pulp and seeds from grape processing waste), or as processing aids when used in the production of bioethanol. Grape berry cell walls are recalcitrant towards degradation, therefore knowledge about their structures and compositions, as well as how the application of enzymes modify these structures is essential in order to optimise these processes. The aim of this study was to extend current knowledge by using a mixture of existing and novel methodologies to study grape berry cell walls by focusing on the profiles of polymers present in the walls. Cell wall profiling techniques used in this study include the Comprehensive Microarray Polymer Profiling (CoMPP) method that employs monoclonal antibodies and Carbohydrate Binding Modules (CBM) which specifically recognise the polymers in the plant cell wall. With this method we measured the abundance of specific polymers and traced the fluctuation in their levels of abundance as influenced by external factors such as enzyme hydrolysis. The CoMPP method was coupled with monosaccharide profile analysis by GC-MS to determine the building blocks of the cell wall polymers, as well as with Infrared Spectroscopy to monitor the changes in the bulk chemistry profile. Data sets generated by the cell wall profiling methods were analysed with uni- and multivariate statistical methods to detect the major patterns in the data. This study highlighted the cell wall differences on the polymer level, in the berry skin cells of Pinotage grapes at different ripeness levels and how it changes during a standard wine fermentation, leading to the release of homogalacturonans and the exposing of arabinogalactan proteins. When maceration enzymes were added, further depectination was evident and the enzymes unravelled the cell wall of the ripe grapes. In overripe grapes no additional degradation could be observed due to maceration enzyme actions, presumably indicating that the endogenous grape enzymes already caused extensive degradation. When purified enzymes were incubated under buffered conditions with isolated skin cell walls from Pinotage grapes or with Chardonnay grape pomace, different levels of enzymatic hydrolysis were observed and defined. The sequence in which cell wall polymers were extracted, and the influence of specific enzymes in facilitating the extraction process, provided important information on the accessibility of specific cell wall polymers. Synergistic action between, for example an endo-polygalacturonase (EPG) and an endo-glucanase (EG) was demonstrated with CoMPP. This EPG and EG synergism was also demonstrated with a yeast strain (a Saccharomyces paradoxus x S. cerevisiae hybrid) fermented in a buffered pomace suspension. This yeast strain has a native EPG and was engineered to also express a recombinant EG from a genome integrated cassette. The cell walls isolated from the pomace after fermentation were unravelled and depectination took place, as evident from CoMPP data. The cell wall profiling techniques used in this study were proven to be fast and sensitive. It provided insights into the structure of grape cell walls and was used to evaluate the changes due to ripening, fermentation, enzymatic hydrolysis and a heat pre-processing treatment. In addition to the knowledge gained, we also demonstrated that these techniques can be used to evaluate different enzymes and enzyme combinations as well as the potential of microorganisms to degrade grape tissue.

AFRIKAANSE OPSOMMING: Maserasie ensieme vir die wynindustrie is ensiem mengsels wat hoofsaaklik pektinases, sellulases en hemisellulases bevat en word tydens wynbereiding gebruik om die druifkorrel se selwand af te breek, die polifenole en aroma molekules vry te stel en sodoende die wyn kwaliteit te verbeter. Hierdie soort ensieme word ook gebruik om inkomste-genererende molekules vanuit druiweprosesserings afval (doppe, pulp en pitte) te isoleer, en ook as prosesserings hulpmiddels in die produksie van bioetanol. Druifkorrel selwande is weerstandig teen ensiem afbraak en daarom is kennis oor die struktuur en samestelling van die selwand, asook hoe die selwand strukture deur die toediening van ensieme verander word noodsaaklik om sodoende hierdie prosesse te optimaliseer. Die doel van hierdie studie was om die huidige kennis uit te brei deur bestaande asook nuwe metodes te gebruik om die druifkorrel selwand te bestudeer met die fokus op die polimeerprofiel van die selwande. Selwand karakteriserings tegnieke wat in hierdie studie gebruik is sluit in die Comprehensive Microarray Polymer Profiling (CoMPP) metode wat monoklonale teenliggaampies en koolhidraat bindende modules (Carbohydrate binding modules, CBMs) wat spesifiek die selwandpolimere van die plant selwand herken, gebruik. Met hierdie metode het ons die vlakke van spesifieke polimere gemeet asook die skommeling in hulle vlakke soos dit beïnvloed is deur eksterne faktore soos ensiem hidroliese. Die CoMPP metode is tesame met monosakkaried profiel analise, met behulp van GC-MS, wat die boublokke van die selwand polimere bepaal, asook infrarooi spektroskopie om die veranderinge in die oorhoofse chemiese profiel te bepaal, gebruik. Datastelle wat met die selwand karakteriserings tegnieke gegenereer is, is ontleed met een- en multiveranderlike statistiese metodes om die hoof tendense in die data op te spoor. Hierdie studie het die selwand verskille, op die polimeervlak, van Pinotage druiwe uitgelig. Verskillende rypheidsgrade asook hoe dit verander tydens ‘n standaard wynfermentasie is gevolg. Laasgenoemde het die vrystelling van homogalakturonaan en die ontbloting van arabinogalaktoproteïene tot gevolg gehad. Met die byvoeging van maserasie ensieme was dit duidelik dat addisionele pektienverwydering plaasgevind het en dat die ensieme die selwand van die ryp druiwe ontrafel het. In oorryp druiwe was daar geen addisionele selwand afbreking sigbaar as gevolg van die aksie van maserasie ensieme nie, wat moontlik aandui dat die inherente druif ensieme reeds uitgebreide selwand afbraak versoorsaak het. Wanneer gesuiwerde ensieme met geïsoleerde selwande van Pinotage druiwedoppe en met Chardonnay druiweprosesserings afval geïnkubeer is onder gebufferde kondisies, is verskillende vlakke van ensiematiese hidroliese waargeneem en geklassifiseer. Die volgorde waarin die selwand polimere geëkstraheer is, asook die invloed van spesifieke ensieme in die bevordering van die ekstraksie proses, het belangrike inligting verskaf oor die toeganglikheid van spesifieke selwand polimere. Sinergistiese aksie tussen, byvoorbeeld ‘n endo-poligalakturonase (EPG) en ‘n endo-glukanase (EG) is geidentifiseer met behulp van die CoMPP data. Hierdie EPG en EG sinergisme is ook geïllustreer met ‘n gisras (‘n Saccharomyces paradoxus x S. cerevisiae hibried) wat in ‘n gebufferde druifprosesserings afval suspensie gefermenteer het. Hierdie gisras het ‘n endogene EPG en is ontwerp om ook ‘n rekombinante EG uit te druk vanaf ‘n genoom geïntegreerde kasset. Die selwande van die druiweprosesserings afval wat na die fermentasie geïsoleer is, was ontrafel en pektienverwydering het plaasgevind, soos bevestig met CoMPP data. In hierdie studie is bewys dat die selwand karakteriserings tegnieke vinnig en sensitief is. Dit het insigte verskaf oor die struktuur van die druifselwand en is gebruik om die veranderinge as gevolg van rypheidsverskille, wynfermentasie, ensiem hidroliese en hitte prosessering te evalueer. Buiten die bydraes tot kennis oor hierdie onderwerpe, is die bruikbaarheid van hierdie tegnieke ook aangetoon, veral in die evaluasie van verskillende ensieme en ensiemkombinasies, asook mikroörganismes vir die afbraak van druifweefsel.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/96848
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