Thesis (MSc)--Stellenbosch University, 2013. / 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.

Thesis (PhD. (Microbiology)) --University of Limpopo, 2013 / The current study investigated four fungal species namely Aspergillus niger FGSC A733, Aspergillus versicolor EF23, Penicillium citrinum AZ01 and Trichoderma harzianum NCGR 0509 for their abilities to produce cellulases and xylanases in submerged and solid state fermentations. Five different substrates (carboxymethyl cellulose, xylan, common thatch grass, wheat bran and Jatropha curcas seed cake) were examined for their potential use as low cost feedstock for fermentation by the fungal species. Aspergillus niger FGSC A733 produced the highest titres of cellulase and xylanase in solid state fermentations using wheat bran as a substrate. However, because of the need to lower the cost of enzyme production, Jatropha seed cake a relatively underutilised oilseed cake was used. Supplementation of the Jatropha seedcake with 10% common thatch grass (Hyperrhenia sp) resulted in a fivefold increase in the levels of xylanase produced. Cellulase production was not affected by this supplementation. Addition of ammonium chloride increased production of xylanase while cellulase production was not affected nitrogen supplementation. Maximum xylanase was produced on Jatropha seed cake at 25 °C after 96 hours while cellulase was maximally produced at 40 °C after 96 hours of solid state fermentations. Peak production of xylanase was obtained at an initial pH of 3 whilst cellulase was maximally produced at an initial pH of 5. The crude xylanase was most active at pH 5 and cellulase at pH 4. The optimum temperature for cellulase activity was 65 °C and that of xylanase was 50 °C. Under optimized conditions, 6087 U/g and 3974 U/g of xylanase and cellulase per gram of substrate used were obtained respectively. The diversity of cellulases was investigated so as to determine the most appropriate enzyme mixture for saccharification of the common thatch grass. Proteins from the four species under investigation were partially purified by affinity chromatography on swollen Avicel. The proteins were analysed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis SDS-PAGE and zymography. Potential cellulase bands from SDS-PAGE were sequenced by mass spectrometry. The basic logical alignment tool (BLAST) and Clustal W were used for matching and identifying the sequences with closely related ones in the databases. The identified proteins from Penicillium citrinum AZ01 and Aspergillus versicolor EF23 were found to closely resemble a catalytic domain of cellobiohydrolase from Trichoderma sp. The three proteins obtained from Aspergillus niger showed resemblance to 1,4-beta glucan cellobiohydrolase A precursor from Aspergillus niger FGSC A733 was also found to have cellobiase and endoglucanase activity was determined using cellobiase and carboxymethyl cellulose as substrates. Cellulase and xylanase zymograms of proteins from A. niger FGSC A733 demonstrated six active bands ranging from 20 kDa to 43 kDa for cellulase and a 31 kDa active band for xylanase. The cellulase produced by Aspergillus niger FGSC A733 on Jatropha seed cake under optimised conditions was used for saccharification of 2% (w/v) common thatch grass (CTG) in combination with Celluclast™. Celluclast™ and Aspergillus niger cellulase were mixed at different ratios and the amount of glucose produced over time was monitored using high performance liquid chromatography (HPLC). A ratio of 2 volumes Celluclast™ to one volume Aspergillus niger cellulase was chosen for the saccharification process. The main enzymes in the mixture were identified using peptide mass fingerprinting as endoglucanases from the Celluclast™ and cellobiase from the Aspergillus niger cellulase. Concentration of the Celluclast™ tenfold times (164 FPU) improved the yield of glucose by 42.8 and 37.8% in acid and alkali pre-treated CTG, respectively. Concentrating Aspergillus niger cellulase (13.2 FPU) decreased the production of glucose by 4.8% in acid pre-treated CTG while in alkali pre-treated CTG, a 5% increase in glucose production was observed. Increasing the substrate loading of acid pre-treated CTG from 2% to 10% (w/v) resulted in a two and a half times increase in glucose production while an increase of 1.5 g/l glucose was obtained from 7% (w/v) alkali pre-treated CTG. Addition of xylanases from Aspergillus niger to the Celluclast™-Aspergillus niger cellulase mixture decreased glucose production by 16.3% on acid pre-treated CTG while there was an increase of 18.3% glucose in alkali pre-treated CTG. Addition of enzyme preparations from Aspergillus versicolor EF23, Penicillium citrium AZ01 and Trichoderma harzianum NCGR 0509 to the Celluclast™-Aspergillus niger cellulase mixture resulted in lower glucose production both in acid and alkali pre-treated CTG. Addition of Pentopan™ improved glucose production by 8 and 25% on 10% acid and 7.5% alkali loading of pre-treated CTG respectively. The optimal conditions for the production of the glucose rich hydrolysate in 10% (w/v) acid and 7% (w/v) alkali pre-treated CTG was found to be the use of Celluclast™-Aspergillus niger cellulase-Pentopan™ mixture (164 FPU Celluclast™ and 13 FPU Aspergillus niger cellulase, 7178 IU) Pentopan™ at 50 °C for 32 hours. The fermentability of the glucose in glucose-rich CTG hydrolysates to ethanol using Saccharomyces cerevisae WBSA 1386 and Candida shehatae CSIR Y-0492 was investigated. The highest yield of ethanol produced by S. cerevisae WBSA 1386 was 9.8 g/l in the alkali pre-treated CTG hydrolysate and 8.7 g/l in acid pre-treated CTG. C. shehatae CSIR Y-0492 produced 9 g/l of ethanol in alkali pre-treated CTG within 48 hours while acid pre-treated CTG hydrolysate produced 8.8 g/l of ethanol within 24 hours of the fermentation process. Addition of the nutrient supplement boosted the ethanol yield in the acid pre-treated hydrolysates. The consumption of glucose during fermentation by S. cerevisae WBSA 1386 and C. shehatae CSIR Y-0492 on average was 97%. The C. shehatae CSIR Y-0492 was expected to produce much higher ethanol yield than the Saccharomyces because of its ability to utilize xylose for ethanol production. This however was not observed in this investigation. The conclusion of this study is that it is possible to produce bioethanol from Hyperrhenia spp. (CTG) using a combination of fungal enzymes for the production of fermentable sugars.

Orientador: Eleonora Cano Carmona / Banca: Aline Aparecida Pizzirani Kleiner / Banca: Helia Hamuri Sato / Banca: Rosa dos Prazeres M.F. Inocentes / Banca: Marcia Regina Brochetto Braga / Resumo: Enzimas degradadoras de xilana, principal componente da hemicelulose, têm sido utilizadas em várias aplicações biotecnológicas, sendo que em alguns processos é necessário o uso de enzimas purificadas. Aplicações comerciais para as enzimas xilanolíticas envolvem a hidrólise enzimática da xilana, que está presente nos resíduos agrícolas e agroindustriais, sendo convertido a xilose e outros açúcares, que podem ser utilizados como substratos em processos fermentativos para a obtenção de proteínas celulares, combustíveis líquidos e outras substâncias químicas. A utilização destas enzimas também diminui a liberação de agentes poluentes em determinados efluentes, como da indústria de polpa de celulose. Xilanases e β- xilosidases são produzidas principalmente por bactérias e fungos, sendo que em geral, os fungos as produzem em níveis mais elevados. O gênero Penicillium apresenta espécies já caracterizadas como boas produtoras destas enzimas. Uma linhagem deste gênero, isolada de solo brasileiro, na região da Mata Atlântica e identificada como Penicillium sclerotiorum destacou-se por produzir xilanase em níveis elevados. O objetivo deste trabalho consistiu na avaliação da influência das condições de cultivo sobre a produção do complexo xilanolítico produzido por P. sclerotiorum, na caracterização físico-química desse sistema, bem como purificação e caracterização bioquímica de seus principais componentes. Por meio da determinação das condições ótimas de produção e da caracterização deste complexo enzimático foi possível estabelecer metodologias eficientes de purificação de xilanases e uma β-xilosidase. Através da caracterização físico-química das enzimas purificadas, foi possível avaliar seu potencial biotecnológico, visando futuras aplicações em processos industriais. / Abstract: Xylan degrading enzymes, the main component of hemicellulose, have been used in various biotechnological applications, and in some cases the use of purified enzymes is necessary. Commercial applications of xylanolytic enzymes involve the enzymatic hydrolysis of xylan, which is present in agricultural and agro-industrial wastes, and can be converted to xylose and other sugars, which can be further used as substrates in fermentation processes to obtaining cellular protein, liquid fuels and other chemicals. The utilization of these enzymes also decreases the release of certain pollutants in wastewater, as in the pulp and paper industry. Xylanases and β-xilosidases are mainly produced by bacteria and fungi, and in general, the fungi produce them at higher levels. The genus Penicillium presents species already characterized as good producers of these enzymes. One strain of this genus isolated from Brazilian soil in the Mata Atlântica region and identified as Penicillium sclerotiorum attracted attention by producing xylanase in high levels. The objective of this study was to evaluate the influence of culture conditions on the production of the xylanolytic complex produced by P. sclerotiorum to characterize physical and chemical properties of this system as well to purify and biochemical characterize its main components. By determining optimal conditions for production and by characterizing this enzymatic complex it was possible to establish efficient methodologies for purification of xylanases and one β-xylosidase. Through their physical and chemical characterization, it was possible to evaluate their biotechnological potential for future applications in industrial processes. / Doutor

Made available in DSpace on 2014-06-11T19:32:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-08-07Bitstream added on 2014-06-13T19:22:45Z : No. of bitstreams: 1 knob_a_dr_rcla.pdf: 1207813 bytes, checksum: 318e70abc84de2d440d3a9b60c7b7088 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Enzimas degradadoras de xilana, principal componente da hemicelulose, têm sido utilizadas em várias aplicações biotecnológicas, sendo que em alguns processos é necessário o uso de enzimas purificadas. Aplicações comerciais para as enzimas xilanolíticas envolvem a hidrólise enzimática da xilana, que está presente nos resíduos agrícolas e agroindustriais, sendo convertido a xilose e outros açúcares, que podem ser utilizados como substratos em processos fermentativos para a obtenção de proteínas celulares, combustíveis líquidos e outras substâncias químicas. A utilização destas enzimas também diminui a liberação de agentes poluentes em determinados efluentes, como da indústria de polpa de celulose. Xilanases e β- xilosidases são produzidas principalmente por bactérias e fungos, sendo que em geral, os fungos as produzem em níveis mais elevados. O gênero Penicillium apresenta espécies já caracterizadas como boas produtoras destas enzimas. Uma linhagem deste gênero, isolada de solo brasileiro, na região da Mata Atlântica e identificada como Penicillium sclerotiorum destacou-se por produzir xilanase em níveis elevados. O objetivo deste trabalho consistiu na avaliação da influência das condições de cultivo sobre a produção do complexo xilanolítico produzido por P. sclerotiorum, na caracterização físico-química desse sistema, bem como purificação e caracterização bioquímica de seus principais componentes. Por meio da determinação das condições ótimas de produção e da caracterização deste complexo enzimático foi possível estabelecer metodologias eficientes de purificação de xilanases e uma β-xilosidase. Através da caracterização físico-química das enzimas purificadas, foi possível avaliar seu potencial biotecnológico, visando futuras aplicações em processos industriais. / Xylan degrading enzymes, the main component of hemicellulose, have been used in various biotechnological applications, and in some cases the use of purified enzymes is necessary. Commercial applications of xylanolytic enzymes involve the enzymatic hydrolysis of xylan, which is present in agricultural and agro-industrial wastes, and can be converted to xylose and other sugars, which can be further used as substrates in fermentation processes to obtaining cellular protein, liquid fuels and other chemicals. The utilization of these enzymes also decreases the release of certain pollutants in wastewater, as in the pulp and paper industry. Xylanases and β-xilosidases are mainly produced by bacteria and fungi, and in general, the fungi produce them at higher levels. The genus Penicillium presents species already characterized as good producers of these enzymes. One strain of this genus isolated from Brazilian soil in the Mata Atlântica region and identified as Penicillium sclerotiorum attracted attention by producing xylanase in high levels. The objective of this study was to evaluate the influence of culture conditions on the production of the xylanolytic complex produced by P. sclerotiorum to characterize physical and chemical properties of this system as well to purify and biochemical characterize its main components. By determining optimal conditions for production and by characterizing this enzymatic complex it was possible to establish efficient methodologies for purification of xylanases and one β-xylosidase. Through their physical and chemical characterization, it was possible to evaluate their biotechnological potential for future applications in industrial processes.

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