Expression and evaluation of enzymes required for the degradation of galactomannan

Malherbe, Alexander Robert (2013-03)

Thesis (MSc)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: The need for a cost-effective and environmentally friendly substitute for fossil fuels has resulted in significant attention to the production of bioethanol. Lignocellulose being the most abundant renewable resource on the planet consists of cellulose, hemicelluloses and lignin. It can be exploited as a source of fermentable sugars for the conversion to ethanol which may serve as the ultimate fossil fuel replacement. Hemicelluloses, contributing one third of lignocellulose, consists of xylan and mannan. Mannan consists of glucomannan, galactomannan and galactoglucomannan. A cocktail of enzymes are required for its complete hydrolysis, including β-mannanase, β-mannosidase, α-galactosidase, β-glucosidase and acetyl-mannan esterases. A need has arisen for the development of a recombinant microorganism capable of converting lignocelluloses to bioethanol through an economically feasible process. The yeast Saccharomyces cerevisiae naturally ferments hexose sugars into ethanol and has been used in various industrial applications due to its robustness in industrial processes, its well-developed expression systems, its frequent use as a model organism for heterologous gene expression and its current GRAS (Generally Regarded As Safe) status. This yeast is unable to naturally utilise complex lignocelluloses. Recombinant biotechnology can be implemented to overcome this limiting factor. Due to certain restraints by the yeast S. cerevisiae such as hyperglycosylation and poor secretion capacity, alternative hosts such as Aspergillus niger has also been considered for heterologous protein production. The Aspergillus aculeatus β-mannanase (man1) and Talaromyces emersonii α-galactosidase (Agal) genes were expressed in S. cerevisiae Y294. The cDNA of A. niger β-mannosidase (cAnmndA) and synthetic Cellvibrio mixtus β-mannosidase (CmMan5A) were expressed in A. niger. The sequence coding for the native secretion signal from CmMan5A was removed and replaced with the XYNSEC sequence (yielding XYNSEC-CmMan5A) and expressed in E. coli DH5α. The recombinant Man1, Agal, cAnmndA, CmMan5A and XYNSEC-CmMan5A displayed optimal pH of 5.47, 2.37, 3.4, 3.4 and 5.47, respectively, and optimal temperatures of 70°C for Man1, Agal, cAnmndA and CmMan5A and 50°C for XYNSEC-CmMan5A. Activity levels of Man1, Agal, cAnmndA, CmMan5A and XYNSEC-CmMan5A peaked at 36.08, 256.83, 11.61, 7.58 and 2.14 nkat/ml, respectively. Co-expression of Agal and man1 led to a decrease in enzyme secretion and therefore individual expression of these genes should be considered rather than co-expression. The enzymatic activity of Man1, Agal and CmMan5A resulted in a significant decrease in the viscosity of galactomannan when used synergistically. This study confirmed successful production of galactomannan hydrolysing enzymes by the yeast S. cerevisiae and the fungus A. niger, as well as providing insight into the synergistic effect of these enzymes on the viscosity of galactomannan.

AFRIKAANSE OPSOMMING: Die behoefte vir 'n koste-effektiewe en omgewingsvriendelike plaasvervanger vir fossielbrandstowwe het tot 'n beduidende belangstelling in die produksie van bio-etanol gelei. Lignosellulose synde die volopste hernubare hulpbron op die planeet bestaan uit sellulose, hemiselluloses en lignien. Dit kan as 'n bron van fermenteerbare suikers vir die omskakeling na etanol benut word, wat kan dien vir uiteindelike fossielbrandstofvervanging. Hemiselluloses, wat bydra tot 'n derde van lignosellulose, bestaan uit xilaan en mannaan. Mannaan bestaan uit glukomannaan, galaktomannaan en galaktoglukomannaan. 'n Mengsel van ensieme word vir die volledige hidroliese van mannaan benodig, insluitende β-mannanase, β-mannosidase, α-galaktosidase, β-glukosidase en asetiel-mannaan esterases. 'n Behoefte bestaan vir die ontwikkeling van 'n rekombinante mikroörganisme wat in staat is tot die omskakeling van lignoselluloses na bio-etanol deur middel van 'n ekonomies lewensvatbare proses. Die gis Saccharomyces cerevisiae kan heksoe suikers na etanol omskakel en word gebruik in verskeie industriële toepassings as gevolg van sy robuustheid in industriële prosesse, goed ontwikkelde uitdrukking sisteme, gereelde gebruik as 'n model-organisme vir heteroloë uitdrukking van gene en huidige GRAS (Generally Regarded As Safe) status. Die gis is nie daartoe in staat om komplekse lignosellulose te benut nie. Rekombinante biotegnologie kan egter geïmplementeer word om hierdie beperkende faktor te oorkom. As gevolg van sekere beperkinge van die gis S. cerevisiae soos hiperglikosilering en lae sekresie kapasiteit, is alternatiewe gashere soos Aspergillus niger ook oorweeg vir heteroloë proteïenproduksie. Die Aspergillus aculeatus β-mannanase (man1) en Talaromyces emersonii α-galaktosidase (Agal) gene is in S. cerevisiae Y294 uitgedruk. Die cDNA van A. niger β-mannosidase (cAnmndA) en sintetiese Cellvibrio mixtus β-mannosidase (CmMan5A) is in A. niger uitgedruk. Die DNA volgorde wat kodeer vir die natuurlike sekresiesein van CmMan5A is verwyder en vervang met die XYNSEC volgorde (gegewe XYNSEC-CmMan5A) en uitgedruk in E. coli DH5α. Die rekombinante Man1, Agal, cAnmndA, CmMan5A en XYNSEC-CmMan5A vertoon optimale pH kondisies van 5.47, 2.37, 3.4, 3.4 en 5.47, onderskeidelik, en die optimale temperatuur van 70°C vir Man1, Agal, cAnmndA en CmMan5A en 50°C vir XYNSEC-CmMan5A. Aktiwiteitsvlakke van Man1, Agal, cAnmndA, CmMan5A en XYNSEC-CmMan5A het 'n maksimum bereik op 36.08, 256.83, 11.61, 7.58 en 2.14 nkat/ml, onderskeidelik. Gesamentlike uitdrukking van Agal en man1 het tot 'n afname in ensiemsekresie gelei en dus moet individuele uitdrukking van hierdie gene eerder as gesamentlike-uitdrukking oorweeg word. Die ensiematiese aktiwiteite van Man1, Agal en CmMan5A het tot 'n beduidende afname in die viskositeit van galaktomannaan gelei wanneer dit sinergisties gebruik word. Hierdie studie bevestig suksesvolle produksie van galaktomannaan hidrolitiese ensieme in die gis S. cerevisiae en die fungus A. niger, en verskaf insig in die sinergistiese effek van hierdie ensieme op die viskositeit van galaktomannaan.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/79817
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