Bioprospecting for beta-glucosidases and beta-xylosidases from non-Saccharomyces yeast

Omardien, Soraya (2013-03)

Thesis (MSc)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: The argument of whether to use food for biofuel (bioethanol) production prompted the search for an alternative non-food biomass, such as lignocellulose, as feedstock for bioethanol production. However, a hindrance in producing bioethanol from lignocellulose on an industrial scale is the cost associated with hydrolysing the lignocellulose to its respective sugar monomers. Improving enzyme production and enhancement of enzyme cocktails for efficient lignocellulose hydrolysis is, therefore, a necessary prerequisite. In this study, a yeast culture collection from the Wine and Fermentation Technology Division (ARC Infruitec- Nietvoorbij, Stellenbosch, South Africa), isolated from fruit from various regions in South Africa, was screened for β-glucosidase and β-xylosidase enzyme activities. β-glucosidases catalyse the hydrolysis of cellobiose and by doing so prevents end-product inhibition of cellobiohydrolases and endoglucanases during cellulose degradation. Similarly, β-xylosidases hydrolyse xylobiose and prevents end-product inhibition of endoxylanases during hemicellulose degradation. After initially screening 2180 non- Saccharomyces yeasts, two yeast isolates were selected that could potentially serve as enzyme source for lignocellulose hydrolysis; one as a producer of a β-glucosidase and another as a β-xylosidase producer. The yeasts were identified as a β-glucosidase producing Rhodotorula slooffiae-like yeast isolate 131B2 and a β-xylosidase producing Aureobasidium pullulans isolate 23B25, respectively. The production of β-glucosidase by Rhodotorula slooffiae-like yeast isolate 131B2 and of β-xylosidase by Aureobasidium pullulans isolate 23B25 was optimised using response surface methodology according to a central composite design. Subsequently, the crude and partially purified enzymes were characterised based on molecular mass, pH optima and stability, temperature optima and stability and inhibition by lignocellulose hydrolysis end-products, such as glucose, xylose and ethanol. The crude β-glucosidase from Rhodotorula slooffiae-like yeast isolate 131B2 was also compared to the commercial Aspergillus niger βglucosidase preparation (Novozyme 188) based on the characteristics mentioned above and as βglucosidase supplement during Avicel (microcrystalline cellulose) hydrolysis by the commercial cellulase preparation (Celluclast). The crude β-xylosidase by Aureobasidium pullulans isolate 23B25 could not be compared to a commercial β-xylosidase as none was available at the time of the study. During the study, the crude β-glucosidase 131B2 and β-xylosidase 23B25 showed potential as lignocellulose hydrolytic enzymes. Attempts were made to obtain the β-glucosidase and β-xylosidase genes from the respective yeast isolates using PCR-based approaches and by constructing cDNA libraries. However, cloning the β-glucosidase and β-xylosidase genes using these methods proved after several attempts to be unsuccessful, although, during this section of the study valuable information was obtained about the obstacles involved with using these approaches when the desired gene sequence is unknown and novel.

AFRIKAANSE OPSOMMING: Die debat oor die toepaslikheid van voedsel vir bio-brandstofproduksie (bio-etanol), het daartoe gelei dat alternatiewe nie-voedsel grondstowwe, soos lignosellulose, as voermateriaal vir bio-ethanol ondersoek word. Die koste geassosieer met die hidrolise van lignosellulose na die onderskeie suiker monomere belemmer industriële-skaal toepassing van lignosellulose vir bio-etanolproduksie. Verbeterde ensiemproduksie en verhoogde doeltreffendheid van ensiemmengsels vir lignosellulose hidrolise is dus ‘n noodsaaklik voorvereiste. In hierdie studie is 'n giskultuurversameling geisoleer vanaf vrugte van verskillende streke in Suid-Afrika deur die Wyn en Fermentasie Tegnologie Afdeling (ARC Infruitec-Nietvoorbij, Stellenbosch, Suid-Afrika) vir β-glukosidase en β-xilosidase ensiemaktiwiteite gesif. β-glukosidases wat die hidrolise van sellobiose kataliseer voorkom eindprodukinhibisie van sellobiohidrolases en endoglukanases tydens sellulose afbraak. β-xilosidases, op hul beurt, hydroliseer xilobiose en voorkom eindprodukinhibisie van endoxilanases tydens hemisellulose afbraak. Na afloop van die aanvanklike sifting van 2180 nie-Saccharomyces giste, is twee giste wat potensiëel as 'n ensiembron vir lignosellulose hidrolise kan dien geselekteer; een vir β-glukosidase en ‘n ander vir β-xilosidase produksie. Die giste is as ʼn β-glukosidase-produserende Rhodotorula slooffiaeagtige gisras 131B2 en ʼn β-xilosidase-produserende Aureobasidium pullulans gisras 23B25 onderskeidelik geïdentifiseer. Die Rhodotorula slooffiae-agtige gisras 131B2 se produksie van β-glukosidase en die Aureobasidium pullulans gisras 23B25 produksie van β-xylosidase was geoptimiseer met behulp van “response surface methodology” volgens 'n “central composite design”. Daarna was die gedeeltelik-gesuiwerde kru-ensieme volgens molekulêre massa, pH optima en stabiliteit, temperatuur optima en stabiliteit, en inhibisie deur lignocelluloses hidrolise end-produkte soos glukose, xylose en etanol, gekarakteriseer. Die kru βglukosidase van die Rhodotorula slooffiae-agtige gisras 131B2 is ook met die kommersiële Aspergillus niger β-glukosidase (Novozyme 188) volgens die eienskappe vroeër genoem vergelyk en as β-glukosidase aanvulling tydens die kommersiële sellulase (Celluclast) se hidrolise van Avicel (mikrokristalline sellulose). Die kru β-xylosidase van die Aureobasidium pullulans gisras 23B25 kon nie vergelyk word met 'n kommersiële β-xylosidase nie, aangesien daar nie een beskikbaar was tydens die studie nie. Gedurende die studie het altwee, die kru β-glukosidase 131B2 en β-xylosidase 23B25, potensiaal getoon as lignosellulose hidrolitiese ensieme. Pogings was aangewend om die β-glukosidase en β-xilosidase gene vanuit die onderskeie gis isolate met behulp van PKR-gebaseerde tegnieke en die opstel van cDNA biblioteke te kloneer. Hierdie klonering strategieë was egter na verskeie pogings onsuksesvol, maar waardevolle inligting oor die struikelblokke betrokke by die gebruik van hierdie benaderings wanneer die gewenste geen se DNS basispaarvolgorde onbekend en uniek is, was verkry.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/80152
This item appears in the following collections: