Xilooligossacarídeos (XOS) são produzidos a partir materiais lignocelulósicos contendo xilanos através de métodos químicos, hidrólise enzimática direta de um substrato susceptível à ação de xilanases e outras enzimas líticas, ou uma combinação de tratamentos químicos e enzimáticos. Os XOS são reconhecidos por trazerem benefício a saúde e são considerados ingredientes prebióticos. A utilização de resíduos agro-industriais produzidos no Rio Grande do Sul, tais como a casca de arroz, a casca de soja e o extrato de malte proveniente de cervejarias tornam-se substratos com grande potencial para a produção da enzima xilanase, principalmente em cultivo em estado sólido. Neste trabalho, foram analisados o potencial de diversos fungos selvagens e de um fungo recombinante na produção da enzima xilanase e outras enzimas importantes para a hidrólise de biomassa lignocelulósica, e a aplicação destes microrganismos para a produção de XOS em cultivos em estado sólido. Através de uma seleção de fungos foi possível definir o Aspergillus brasiliensis BLf1 como maior produtor de xilanase em cultivo sobre casca de arroz, obtendo atividade de 120,5 U.g-1 substrato. Nesta seleção foram analisadas também as enzimas celulase, β-glicosidase e β-xilosidase Um planejamento experimental fracionário 2(5-1) deste fungo selecionado e do fungo recombinante Aspergillus nidulans XynC A773 determinaram variáveis que influenciam a produção da enzima xilanase, chegando a uma atividade máxima de 230,7 U.g-1 para A. brasiliensis BLf1 e 187,9 U.g-1 para A. nidulans XynC A773. Posteriormente, estas preparações enzimáticas foram aplicadas à casca de arroz para hidrolisar sua estrutura hemicelulósica polimérica e obter xilolossacarídeos (37,25 mg de XOS.g-1 de substrato e 75,92 mg de XOS.g-1 de substrato, respectivamente). Por fim, foi avaliado o potencial prebiótico de XOS obtidos. Os resultados deste estudo revelaram o crescimento de L. plantarum BL011 e B. lactis BB-12 em XOS, com um aumento de massa celular seca de até 1,7 g.L-1 em 120h. Os resultados obtidos neste trabalho sugerem que é possível a obtenção de XOS a partir de resíduos abundantes no Estado e que os mesmos possuem potencial para serem utilizados como prebióticos, podendo, portanto, ser usados em aplicações relacionadas aos alimentos funcionais. / Xylooligosaccharides (XOS) are produced from lignocellulosic materials containing xylan by chemical methods, direct enzymatic hydrolysis of susceptible substrates using xylanases and other lytic enzymes, or a combination of chemical and enzymatic treatments. XOS are recognized for bringing benefit to health of the host and are considered prebiotic ingredients. The use of lignocellulosic residues produced in Rio Grande do Sul, such as rice husk, soybean hulls, and spent malt from brewery hold potential for the production of xylanase enzyme, especially under solid-state cultivation. In this work, several wild strains of fungi and one recombinant strain were tested for their potential of producing xylanase and their application in solid-state cultivation to obtain XOS. It was possible to define the Aspergillus brasiliensis BLf1 as the best producer of xylanase on rice husk, obtaining an activity of 120.5 U.g-1. Other important lytic enzymes were also analyzed: cellulase, β-glucosidase, and β-xylosidase The statistical experiment fractional factorial design 2(5-1) of cultures of this fungus and of the recombinant strain Aspergillus nidulans XynC A773 defined the variables that influenced the production of xylanase, showing maximal activities of 230.7 U.g-1 for A. brasiliensis BLf1 and 187.9 U.g-1 for A. nidulans XynC A773. Subsequently, these enzymatic preparations were applied to rice husk to hydrolyse its polymeric hemicellulosic structure and obtain xylooligosaccharides (37.25 mg XOS.g-1 substrate and 75.92 mg XOS.g-1 substrate, respectively). Finally, the prebiotic potential of XOS was evaluated by using them to grow L. plantarum BL011 and B. lactis BB-12, showing an increase in the dry cell mass of 1.7 g.L-1 at 120 hours. The results obtained in this research suggest that it is possible to obtain XOS from agro industrial residues of local production and they have the potential to be used as prebiotics, and could be used in food related applications.

Orientador: Rubens Monti / Banca: Rubens Monti / Banca: Luis Henrique Souza Guimarães / Banca: Olga Luisa Tavano / Banca: Maristela de Freitas Sanches Peres / Banca: Luis Vitor Silva do Sacramento / Resumo: A descoberta de resíduos agrícolas, como fonte de energia renovável tem colaborado para o desenvolvimento industrial e preservação do meio ambiente. Desta maneira, a degradação da parede celular destes resíduos tem grande importância na fabricação de pães, alimentos, bebidas, têxtil, papel e celulose entre outros produtos. A degradação enzimática destes polímeros esta se tornando uma alternativa mais atraente do que a utilização de substâncias químicas e processos mecânicos. Alguns fungos termófilos participam desta degradação enzimática, como Humicola grisea var. thermoidea, isolado do solo, é conhecido como bom produtor do complexo xilanolítico. Além deste, um fungo termófilo isolado da polpa do fruto cupuaçuzeiro em decomposição foi estudado e produziu xilanase. Esses fungos foram mantidos em meios sólidos contendo ágar e farinha de aveia. O fungo isolado da polpa do fruto cresceu em meio liquido empregando diferentes resíduos agrícolas como fonte de carbono. Dentre os vários resíduos empregados para se otimizar a produção de xilanase, o melhor foi o sabugo de milho. Esse foi utilizado nos experimentos na proporção de 1,0% (m/v), em meio líquido contendo 0,1% CaCO3, 0,5% NaCl, 0,1% NH4Cl; 0,5% água de maceração de milho. O pH ótimo foi 5,0 e a temperatura ótima de 60ºC. A xilanase presente no extrato clarificado em caulim apresentou cinética Michaeliana com Km de 10,41 ± 0,282 mgmL-1 e Vmax 3,32 ± 0,053 U (mg protein)-1. Para acompanhar o processo de clarificação da enzima solúvel realizou-se SDS-PAGE verificando a presença de uma banda protéica com atividade xilanolítica de massa molecular de aproximadamente 30kDa e a Cromatografia Sílica Gel P60 para verificar os produtos da hidrólise da xilana em função do tempo e da associação de xilosidase do fungo termófilo Humicola com a xilanase do fungo isolado do fruto... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The researches of agricultural residues as renewable energy sources have been contributing for the industrial development and the environment conservation. Thus, the degradation of these residues' cell walls has great importance in fabrication of bread, food, drinks, textile, paper, cellulose, and other products. The enzymatic degradation of such polymers is becoming an attractive option when compared to the use of chemical substances and mechanical processes. Some thermophilic fungi take role in this enzymatic degradation, such as Humicola grisea var. thermoidea, isolated from soil, and is known to be a good producer of the xylanolytic complex. Besides it, another thermophilic fungus, isolated from the Theobroma grandiflorum decomposing fruit pulp, was studied and also produced xylanases. These fungi were kept in solid media containing agar and oat bran. The fungus isolated from the fruit pulp grew in liquid media in wich different kinds of agricultural residues were employed as carbon sources. Among the various types of residues that were used to optimize the xylanase production, corn cobs proved to be the best one. It was used in assays in a proportion of 1,0% (m/v), in liquid media containing 0.1% of CaCO3, 0.5% of NaCl, 0.1% of NH4Cl, and 0.5% of corn steep liquor. The pH optima was 5.0 and the temperature optima was 60ºC. The xylanase present in the kaolin clarified extract showed a Km of 10.41 ± 0.282 mg.mL-1, and Vmax 3.32 ± 0.053 U (mg.protein)-1. In order to follow the soluble enzyme clarification process, SDS-PAGE was run, verifying the presence of a proteic band with xylanolytic activity of, approximately, 30kDa of molecular mass. Furthermore, Silica Gel 60 Thin Layer Chromatography (TLC) was used to verify the xylan hydrolysis products in function of time and the association of Humicola sp. thermophylic fungus xylosidase with the xylanase produced by the fruit pulp... (Complete abstract click electronic access below) / Doutor

Thesis (MSc. (Biochemistry)) --University of Limpopo, 2016 / The ever increasing cost of fossil-based fuels and the accompanying concerns about their impact on the environment is driving research towards clean and renewable sources of energy. Bioethanol has the potential to be a replacement for liquid transportation fuels. In addition to its near zero nett carbon dioxide emissions, bio-ethanol has a high energy to weight ratio and can easily be stored in high volumes. To produce bioethanol at economically competitive prices, the major cost in the production process needs to be addressed. The addition of enzymes to hydrolyse the lignocellulosic fraction of the agricultural waste to simple sugars is considered to be the major contributor to high production cost. A consolidated bioprocess (CBP) which ideally combines all the steps that are currently accomplished in different reactors by different microorganisms into a single process step would be a more economically feasible solution. In this study the potential of yeast hybridization with a CBP approach was used. In order to evaluate the reduction or elimination of the addition of cellulolytic and hemi-cellulolytic enzymes to the ethanol production process. High cellobiohydrolase I secreting progeny from hybridization of an industrial bioethanol yeast strain, S. cerevisiae M0341, and a laboratory strain S. cerevisiae Y294 were isolated. In order to determine if this characteristic was specific to cellobiohydrolase I secretion, these strains were evaluated for their ability to secrete other relevant recombinant hydrolase enzymes for CBP-based ethanol production. A total of seven S. cerevisiae strains were chosen from a progeny pool of 28 supersecreting hybrids and reconstructed to create two parental strains; S. cerevisiae M0341 and S. cerevisiae Y294, together with their hybrid segregants strains H3M1, H3M28, H3H29, H3K27 and H3O23. Three episomal plasmids namely pNS201, pNS202 and pNS203 were constructed; these plasmids together with two already available plasmids, namely pRDH166 and pRDH182 contained genes for different reporter enzymes, namely β-glucosidase I, xylanase II, endoglucanase lll, cellobiohydrolase l and α-glucuronidase. To allow for selection of the episomal plasmids, homologous recombination was used to replace the functional URA3 gene of selected strains, with the non-functional ura3 allele from the Y294 strain. Enzyme activity was used as an indicator of the amount of enzyme secreted. Fermentation studies in a bioreactor were used to determine the metabolic burden imposed on the segregants expressing the cellobiohydrolase at high levels. In addition all segregants were tested for resistance to inhibitors commonly found in pre-treated lignocellulosic material. The M28_Cel7A was found to be the best secretor of Cel7A (Cellobiohydrolase l); however it seems as though this phenomenon imposes a significant metabolic burden on the yeast. The supersecreting hybrid strains cannot tolerate lignocellulosic inhibitors at concentrations commonly produced during pretreatment / The National Research Foundation - Renewable Energy Scholarship (NRF-RSES)

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19194号 / 農博第2133号 / 新制||農||1034(附属図書館) / 学位論文||H27||N4940(農学部図書室) / 32186 / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 髙野 俊幸, 教授 西尾 嘉之, 教授 梅澤 俊明 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Past literature has supported the supplementation of 25-OHD3 into poultry diets to reduce leg issues and improve muscle accretion. In addition, exogenous feed enzymes are included into poultry diets to increase nutrient utilization. The first objective of this thesis was to determine the effects of different sources and levels 25-OHD3 supplementation on D0-53 Ross x Ross 708 male broiler performance, processing yield, tibia ash, serum Ca and 25-OHD3 status. Data demonstrated that 25-OHD3 supplementation into diets with Low VitD3 (165 IU/kg) can improve broiler performance compared to High VitD3 (2756 IU/kg). The second objective was to investigate the effects of varying phytase inclusion with different xylanase levels on D0-56 Ross x Ross 708 male broiler performance, processing yield and tibia ash. Data exhibited some performance benefit (D46 and D56) when utilizing phytase (1000 and 1500 FTU/kg) with 1500 EPU/kg xylanase and phytase (250 and 1500 FTU/kg) with 3000 EPU/kg xylanase.

No description available.

Le Rovabio Excel ™ est un cocktail enzymatique complexe sécrété par le champignon filamenteux Penicillium funiculosum. La société ADISSEO commercialise cet additif alimentaire destiné à la nutrition animale car les principales enzymes qui le constituent dégradent les polymères contenus dans les céréales, tels que les polysaccharides non amylacés. Ainsi, le Rovabio Excel™ permet d’améliorer la digestibilité et d’augmenter la valeur nutritionnelle des matières premières agricoles en réduisant la viscosité du bol alimentaire des animaux. Dans le but d’augmenter sa compétitivité, ADISSEO a fait conduire des études sur cette solution pour la caractériser biochimiquement et optimiser son potentiel xylanolytique.Ces travaux de thèse s’inscrivent dans ces projets industriels et ont poursuivi deux objectifs distincts. Le premier correspondait à l’augmentation de la thermostabilité de la protéine XynB du Rovabio Excel™, pour lui permettre de résister à la granulation. Le second concernait XynA, la protéine majoritaire de la solution multienzymatique, qui a été caractérisée biochimiquement. Les premiers résultats de caractérisation biochimique de XynA ont montré que la protéine était 100 fois plus active sur β-1,4-glucane que sur xylane. Des tests complémentaires sur pNP-cellobiose et pNP-β-D-Lactopyranose ont révélé que XynA était 5,2 fois plus active sur pNP-cellobiose et possédait une activité « exo ». Enfin, l’analyse des produits d’hydrolyse d’oligosaccharides composés de 2 à 5 unités de glucose a confirmé que la protéine XynA était une cellobiohydrolase de type I, très sensible à l’inhibition par le cellobiose (IC50 - C2 = 17,7 µM). L’étude la thermostabilité de XynB a confirmé que cette protéine n’était pas naturellement thermostable. Les résultats des travaux d’ingénierie avec l’ajout d’un pont disulfure pour rigidifier la structure 3D de la protéine n’ont pas été probants. En revanche, la création de protéines chimères à partir de protéines plus thermostables (TfxA de Thermomonospora fusca et XynII de Trichoderma reesei) a permis d’améliorer la stabilité thermodynamique de XynB avec des Tm augmentés de plus de 10°C / The Rovabio Excel™ is a complex enzymatic cocktail secreted by the filamentous fungus Penicillium funiculosum. The ADISSEO company sells it as food additive for animal feed because the main enzymes degrade polymers contained in grains, such as non-starch polysaccharides. Thus, the Rovabio Excel™ improves the digestibility and increases the nutritional value of agricultural raw materials by reducing the viscosity of the diet of animals. In order to increase its competitiveness, ADISSEO did conduct studies on this solution to characterize it biochemically and maximize its xylanolytic potential.This thesis takes part of this industrial project and have pursued two distinct objectives. The first corresponds to the increase in the thermostability of the protein XynB from the Rovabio Excel™, to enable it to resist at the granulation process. The second was XynA, the major protein of the multienzyme solution, which was characterized biochemically.Initial results of biochemical characterization of XynA showed that the protein was 100 times more active on β-1,4-glucan on xylan. Additional tests on pNP-cellobiose and pNP-β-D-Lactopyranose revealed that XynA was 5.2 times more active on pNP-cellobiose and possess an "exo-acting" activity. Finally, the analysis of products from oligosaccharides hydrolysis, composed of 2 to 5 units of glucose, confirmed that the protein XynA was a type I cellobiohydrolase, very sensitive to inhibition by cellobiose (IC50-C2 = 17.7 µM).The thermostability of XynB study has confirmed that this protein was not thermostable naturally. The results of the engineering work with the addition of a disulfide bridge to rigidify the 3D structure of the protein were not conclusive. However, the creation of chimeric proteins with more thermostable proteins (TfxA from Thermomonospora fusca and XynII from Trichoderma reesei) has improved the thermodynamic stability of XynB with Tm increased by more than 10°C

La création de nouvelles enzymes pour l’hydrolyse de la biomasse est une stratégie clé pour ledéveloppement du bioraffinage. Dans ce contexte, les xylanases de la famille GH11 sont déjàdéployées dans de nombreux procédés industriels et donc bien positionnées pour jouer un rôleimportant dans ces procédés. La cible de cette étude, la xylanase GH11 (Tx-Xyl) de la bactérieThermobacillus xylanilyticus, est une enzyme thermostable et donc une bonne candidate pour destravaux d’ingénierie visant l’amélioration de son activité sur des substrats ligno-cellulosiques.Dans cette étude, deux stratégies d’ingénierie des enzymes ont été employées afin d’obtenir denouvelles informations portants sur les relations structure-fonction au sein de Tx-Xyl. La premièrestratégie a consisté en l’utilisation d’une approche de mutagenèse aléatoire, couplée à l’emploi deméthodes de recombinaison in vitro. Ces travaux avaient pour objectif d’améliorer la capacitéhydrolytique de Tx-Xyl sur la paille de blé. La deuxième stratégie mise en oeuvre s’est appuyée surune approche semi-rationnelle visant la création d’une enzyme chimérique, qui bénéficierait d’uneamélioration des interactions enzyme-substrat au niveau du sous-site -3.Le premier résultat majeur de cette thèse concerne le développement d’une méthode de criblagequi permet l’analyse à haut débit de banques de mutants pour la détection de variants quiprésentent une activité hydrolytique accrue directement sur paille de blé. A l’aide de ce crible, nousavons pu analyser plusieurs banques de mutants, représentant un total de six générations demutants, et identifier une série de combinaisons de mutations différentes. D’un côté, un variant,comportant deux mutations silencieuses, permet une meilleure expression de Tx-Xyl, alors qued’autres enzymes mutées présentent des modifications intrinsèques de leurs aptitudes catalytiques.Comparés à l’enzyme parentale Tx-Xyl, certains mutants solubilisent davantage les arabinoxylanes dela paille et, lorsqu’ils sont déployés avec un cocktail de cellulases, participent à une réactionsynergique qui permet un accroissement du rendement des pentoses et du glucose libérés.A l’aide d’une approche semi-rationnelle, une séquence de 17 acides aminés en provenance d’unexylanase GH11 fongique a été ajoutée à l’extrémité N-terminale de Tx-Xyl, afin de créer de nouveauxbrins β. L’enzyme chimérique a pu être exprimée avec succès et caractérisée. Néanmoins, l’analysede ses propriétés catalytiques a révélé que celle-ci ne présente pas davantage d’interactions avec sonsubstrat dans le sous-site -3, mais les résultats obtenus fournissent de nombreux renseignements surles relations structure-fonction au sein de l’enzyme. De plus, ces travaux nous permettent depostuler que Tx-Xyl posséderait un site de fixation secondaire pour les xylanes, un élement jusqu’iciinsoupçonné dans cette enzyme. Par ailleurs, l’analyse de nos résultats nous permet de proposer uneexplication rationnelle pour l’échec de notre stratégie initiale / Engineering new and powerful enzymes for biomass hydrolysis is one area that will facilitate thefuture development of biorefining. In this respect, xylanases from family GH11 are already importantindustrial biocatalysts that can contribute to 2nd generation biorefining. The target of this study, theGH11 xylanase (Tx-Xyl) from Thermobacillus xylanilyticus is thermostable, and is thus an interestingtarget for enzyme engineering, aiming at increasing its specific activity on lignocellulosic biomass,such as wheat straw. Nevertheless, the action of xylanases on complex biomass is not yet wellunderstood, and thus the use of a rational engineering approach is not really feasible.In this doctoral study, to gain new insight into structure-function relationships, two enzymeengineering strategies have been deployed. The first concerns the development of a randommutagenesis and in vitro DNA shuffling approach, which was used in order to improve the hydrolyticpotency of Tx-Xyl on wheat straw, while the second strategy consisted in the creation of a chimericenzyme, with the aim of probing and improving -3 subsite binding, and ultimately improvinghydrolytic activity.The first key results that has been obtained is the development of a novel high-throughputscreening method, which was devised in order to reliably pinpoint mutants that can better hydrolyzewheat straw. Using this screening method, several generations of mutant libraries have beenanalyzed and a series of improved enzyme variants have been identified. One mutant, bearing silentmutations, actually leads to higher gene expression, while others have intrinsically altered catalyticproperties. Testing of mutants has shown that some of the enzyme variants can improve thesolubilization of wheat straw arabinoxylans and can work in synergy with cellulose cocktails torelease both pentose sugars and glucose.Using a semi-rational approach, 17 amino acids have been added to the N-terminal of Tx-Xyl, withthe aim of adding two extra β-strands coming from a GH11 fungal xylanase. A chimeric enzyme hasbeen successfully expressed and purified and its catalytic properties have been investigated.Although this approach has failed to create increased -3 subsite binding, the data presented revealsimportant information on structure-function relationships and suggest that Tx-Xyl may possess ahitherto unknown secondary substrate binding site. Moreover, a rational explanation for the failureof the original strategy is proposed.

Thesis (PhD (Science) (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Wine fermentation with a yeast strain able to degrade grape cell polysaccharides can result in improved processability and an increase in wine quality by improving extraction of essential compounds from the grapes during the maceration stage. Pectin is the only important cell wall polysaccharide that can be degraded by wild-type Saccharomyces cerevisiae strains. Pectin is degraded by a polygalacturonase (PG) encoded by the PGU1 gene (ORF YJR153W). Only certain S. cerevisiae strains can degrade pectin and PG activity is thus strain specific. The lack of activity in certain strains has been attributed to a number of factors: (1) the complete absence of the PGU1 gene, (2) the PGU1 gene is present but the allele is dysfunctional and (3) the PGU1 gene is present but not transcribed. The lack in transcription has been shown to be due to the gene having a dysfunctional promoter or to regulatory differences between strains. Results published in the literature are contradictory. The primary aim of this investigation was to clarify the regulation of PG activity in S. cerevisiae and to determine why there are differences in PG activity between different strains. Regulation of PG activity between several wine and laboratory strains with varying PG activities was compared by looking at the sequence of the PGU1 gene and its promoter as well as transcription levels of this gene and its main transcription factors, TEC1 and STE12. In order to identify regulatory factors influencing PG activity, the S. cerevisiae genome was screened for activators and inhibitors of PG activity. Fourteen inhibitors and two activators of PG activity were identified during this screen. Real-time PCR analysis showed that the PG activity is regulated by transcription of the PGU1 gene. A linear relationship was demonstrated between PGU1 and its two transcription factors TEC1 and STE12. Some of the genes identified as inhibitors of PGU1 transcription are involved in gene silencing by Telomere Position Effect (TPE) indicating that PGU1 is possibly silenced due to its subtelomeric location within 25 kb from the right telomere of chromosome X. Moving the PGU1 gene with its native regulatory machinery to a different position away from its telomere resulted in an increase in PGU1 transcription and PG activity, demonstrating the epigenetic influence on PGU1 regulation. Results from this study suggested that the strain related difference in PGU1 expression occurs at an epigenetic level, with steric hindrance preventing RNA polymerase access to the PGU1 promoter and thus inhibiting transcription of this gene in some strains. Understanding regulation of PG activity can potentially lead to the development of more effective strategies to improve PG degradation by S. cerevisiae. The genetic model describing regulation of PGU1 transcription was extended by this study and a novel mechanism of regulation of PG activity was identified. The secondary aim of this study written as an addendum to this thesis, focussed on degradation of another grape cell wall polysaccharide xylan by recombinant strains of S. cerevisiae. These strains were enabled to degrade this polysaccharide through heterologous expression of novel xylanase encoding genes from various origins. Xylanase activity of the recombinant strains generated was compared. Overexpressing the complete gene xynA of Ruminococcus flavefaciens, the functional domain xynAa or the functional domain xynAc within optimal conditions for these enzymes all conferred very low xylanase activity to S. cerevisiae, with xynAc resulting in the highest xylanase activity. Since overexpression of the R. flavefaciens xynA gene yielded very low activity under optimal conditions activity in wine making conditions would be negligible. The genes XYN2 and XYN4 from Trichoderma reesei and Aspergillus niger respectively yielded higher levels of activity. According to these results, only the expression of XYN2 and XYN4 could have a potential effect on wine An effective strategy for improving pectin degradation can in future potentially be combined with heterologous expression of a xylanase encoding gene in S. cerevisiae in order to engineer a wine yeast strain with improved polysaccharase abilities. / AFRIKAANSE OPSOMMING: Gisting van druiwe met polisakkaried-afbrekende gisrasse kan lei tot ‘n verbetering in wyn prosessering en tot die produksie van hoër kwaliteit wyne deur die ekstraksie van belangrike wynkomponente uit druifselle te verbeter. Pektien is die hoof komponent van die druifselwand wat deur wilde tipe Saccharomyces cerevisiae giste afgebreek kan word en word afgebreek deur ‘n poligalaktoronase (PG) wat deur die PGU1 (YJR153W) geen gekodeer word. Slegs spesifieke gisrasse kan pektien afbreek en die ensiem aktiwiteit is dus ras-spesifiek. Die gebrek aan PG aktiwiteit in sekere rasse is al omskryf as gevolg van die afwesigheid van die geen, die teenwoordigheid van ‘n nie-funksionele alleel of dat die geen wat teenwoordig is nie uitgedruk word nie. Transkripsie is al bewys om nie plaas te vind nie a.g.v. die teenwoordigheid van ‘n nie-funksionele promotor of a.g.v. ‘n verskil in regulering van transkripsie tussen rasse. Sommige studies wat PG regulering ondersoek het, het teenstrydige resultate verkry. Die hoofdoel van hierdie studie was om PG regulering te ondersoek en te bepaal waarom daar verskille in PG aktiwiteit tussen verskillende gisrasse voorkom. Regulering van PG aktiwiteit is ondersoek tussen wyn en laboratorium gisrasse met wisselende vlakke van PG aktiwiteit deur die DNS volgorde van die PGU1 geen en sy promotor, so wel as die DNS volgorde van die geen se hoof transkripsie faktore TEC1 en STE12 te bepaal. Om reguleerders van PG aktiwiteit te identifiseer is die genoom van die gis S. cerevisiae ondersoek om faktore te identifiseer wat PG aktiwiteit aktiveer of inhibeer. “Real-time PCR” het bewys dat PG aktiwiteit gereguleer word deur transkripsie van die PGU1 geen en dat daar ‘n lineêre verhouding tussen die transkripsie van die PGU1 geen en sy twee hoof transkripsie faktore TEC1 en STE12 bestaan. Sommige van die gene wat geïdentifiseer is as inhibeerders van PG aktiwiteit is voorheen bewys om betrokke te wees by die inhibering van transkripsie deur middel van die telomeer posisie effek, dit dui daarop dat transkripsie van die PGU1 geen moontlik geïnhibeer word as gevolg van die geen se subtelomeriese posisie binne 25 kb vanaf die regter telomeer van chromosoom X. Die PGU1 geen is met sy natuurlike regulerings elemente na ‘n ander posisie in die genoom, weg van sy naaste telomeer geskuif, die verandering in posisie van die geen het gelei tot ‘n toename in PG aktiwiteit en transkripsie van die PGU1 geen en het dus bewys regulering word beïnvloed deur ‘n epigenetiese effek. Die resultate van hierdie studie het daarop gedui dat die verskil in transkripsie van die PGU1 geen plaasvind op ‘n epigenetiese vlak waartydens die chromatien struktuur toegang van die RNA polimerase tot die PGU1 geen voorkom en dus word transkripsie van die geen sodoende in sommige rasse voorkom. Die tweede doelwit van hierdie studie het gefokus op die afbraak van ‘n ander komponent van die druif selwand, xilaan, deur S. cerevisiae. Hierdie navorsing vorm ‘n addendum aan die tesis en Xylanase aktiwiteit van verskeie rekombinante rasse is in hierdie studie vergelyk. Baie lae xylanase aktiwiteit is verleen aan rekombinante giste wat die volledige xynA geen gekloneer van die bakteriee Ruminococcus flavefaciens, asook twee aktiewe domeins van die geen, domein xynAa en domein xynAc uitdruk. Van die voorafgenoemde giste het die uitdrukking van die domein xynAc die rekombinante gis ras met die hoogste aktiwiteit tot gevolg gehad. Ooruitdrukking van die gene XYN2 en XYN4 wat gekloneer is van die fungi Trichoderma reesei en Aspergillus niger onderskeidelik, het beide gisrasse wat oor hoë vlakke van xylanase aktiwiteit beskik tot gevolg gehad. Hierdie resultate dui dus daarop dat van die gene ondersoek in die studie, slegs XYN2 en XYN4 potensiaal het om xylanase aktiwiteit van wyngiste te verbeter. Deur die regulering van PG aktiwiteit te bestudeer kan meer effektiewe strategieë potensieel ontwikkel word om PG aktiwiteit in S. cerevisiae te verbeter. Hierdie studie het die genetiese model wat PG regulering omskryf uitgebrei deur ‘n nuwe meganisme van regulering van toepassing op PGU1 te identifiseer. As ons die regulering van die PGU1 goed verstaan kan dit in die toekoms gekombineer word met ‘n effektiewe strategie om ‘n gis aan te pas om xylaan af te breek, om sodoende ‘n wyngis geneties te verbeter om beide xylaan en pektien te kan afbreek.

Une approche de métagénomique fonctionnelle a été mise en œuvre afin d’étudier les arsenaux enzymatiques produits par les microbiotes intestinaux de termites phytophages et d’identifier de nouvelles enzymes impliquées dans l’hydrolyse des polysaccharides végétaux, notamment des hétéroxylanes. Le criblage à haut débit des banques métagénomiques constituées à partir de trois espèces de termites sur une gamme de substrats chromogéniques a permis d’identifier plusieurs centaines de clones à activité dépolymérisante (glucanase, xylanase, mannanase, arabinanase), ainsi que des clones exprimant des activités auxiliaires (α-L-arabinofuranosidases, β-D-xylosidases, cellobiose hydrolases). Un total de 42 clones métagénomiques a été séquencé, générant 1,5 Mpb d’ADN assemblé en 58 séquences contigües d’une taille moyenne de 37,8 Kbp. 63 nouvelles Glycoside Hydrolases (GH) ont été identifiées. Ces dernières représentent 19 familles de la classification CAZy, dont les familles GH3, GH8, GH10, GH11, GH43 et GH51. Enfin, huit nouvelles enzymes des familles GH43 et GH51 ont été produites chez E. coli et leurs propriétés biochimiques ont été étudiées. Ces enzymes présentent des activités α-L-arabinofuranosidase, β-D-xylosidase ou L-arabinanase / A functional metagenomics approach was used to reveal the enzymatic diversity present in the guts of biomass-feeding termites and to identify enzymes involved in the degradation of biomass components, notably heteroxylans. High-throughput screening of metagenomic libraries, created using three different termite species, was performed using a variety of chromogenic substrates. This allowed the discovery of hundreds of clones expressing targeted biomass-degrading activities (e.g. depolymerases such as glucanase, xylanase, mannanase arabinanase and auxiliary activities such as α-L-arabinofuranosidases, β-D-xylosidases and cellobiohydrolases). A total of 42 clones were selected for a DNA sequence analysis, thus generating 1.5 Mbp that were assembled into 58 contiguous sequences. 63 new Glycoside Hydrolases (GH) belonging to 19 different families of the CAZy classification were identified, including ones from families GH3, GH8, GH10, GH11, GH43 and GH51. Finally, eight new enzymes, from families GH43 and GH51, were produced in E. coli and their biochemical properties were studied. These enzymes display α-L-arabinofuranosidase, β-D-xylosidase or arabinanase activities