O Penicillium echinulatwn linhagem 9A02Sl é um fungo filamentoso que apresenta um sistema celulolítico com potencial para aplicaqão em processos de degradação de materiais lignocelulósicos para produção de etanol. O crescente interesse nesse combustível e a abundância de materiais lignocelulósicos que podem ser usados como matéria-prima fez aumentar o interesse no estudo de celulases. Neste estudo, a linhagem 9A02Sl de Penicillium echinulatum foi cultivada em cultivos submersos em frascos mantidos sob agitação recíproca, com variações quanto às fontes de carbono. Além de crescimento, foram avaliadas as produções de celulases, ~-glicosidases e xilanases e a expressão das enzimas através ale zimogramas em géis de poliacrilamida para detemlinação da massa molecular. Observou-se que o crescimento micelial provocou a redução do pH do meio de cultivo, e que não está relacionado a produção de enzimas. A celulose apresentou-se como indutora para todas as enzimas analisadas. A carboximetilcelulose mostrou-se uma eficiente fonte de carbono para a produção de atividade sobre papel filtro, endoglicanases e xilanases, apesar do baixo crescimento micelial. Celobiose, glicerol e glicose estimularam a produção de ~glicosidases. Uma banda de atividade endoglicanêlsica de 74 kDa foi detectada nos zimogramas de todos os caldos enzimáticas obtidos na presença d1e diferentes fontes de carbono, sugerindo esta seja uma enzima constitutiva. A expressão da ~-glicosidase oconeu ao fmal do cultivo (5° e 6° dias), sendo que em todos os cultivos avaliados houve a expressão de uma banda de 220 kDa, indicando tratar-se de uma enzima constitutiva. A expressão de outras bandas com diferentes massas moleculares sugerem que diferentes genes, fotmats multiméricas ou modificações pós-traducionais estão envolvidos no perfil destas enzimas em Peni'cillium echinulatum. / Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq / The st:rain of Penicillium echinulatum 9A02Sl is a filamentous fungus that presents a cellulolytic system with potential application in processes of degradation of lignocellulosic materiais for ethanol production. The growing interest in fhel and the abundance of lignocellulosic materiais that can be used as raw material has inc:reased the interest in cellulases. In this study, the strain P echinulatum 9A02Sl was grown in submerged cultivation in agitated flasks in presence of different carbon sources. In addition to growth, it was evaluated the production of cellula.ses, Pglucosidases and xylanases, and enzyme expres:sion in activity polyacrylamide gels in order to detemlinate the molecular ma.ss. The mycelial growth decreased pH o f the medium and this fact was not related to enzyme production. The cellulose was an inducer for all the enzymes ana.lyzed. The carboximetilcellulose was found to be an effi[cient carbon source for production o f filter paper a.ctivity, endogluca.nases and xylanases, despite the low mycelial growth. Cellobiose, glycerol and glucose stimulated the production of P-glucosidases. An endoglucanase band of 74 kDa was detected in zymograms o f all enzyme broths obtained in the presence o f different carbon sources, suggesting it is a constitutive enzyme. The expression of P-glucosidase occuned at the end of cultivation (5 and 6 days), and in all medium that was evaluated was observed a 250 kDa band, indica.ting that this is a. constitutive enzyme. The expression of other bands with different molecular mass suggest that different genes, multimeric fonns or post-translational modifications are involved in the expression ofthese enzymes in P echinulatum.

A thesis submitted in fulfillment of the requirement for the Degree Master of Technology: Biotechnology, M.L. Sultan technikon, 2000. / The ever-increasing demand for a wide variety of paper products has led to the pulp and paper industry becoming one of the largest industries in the world. In 1988 the United States alone produced almost 71 million metric tonnes of paper and pulp board (Jeffries, 1992). South Africa has also become one of the major international producers of pulp and paper products. Since 1970, the production of paper and board by the South African industry achieved an average growth rate of 5.2% per annum, and in 1997 South Africa was the twelfth largest producer of pulp and 24th biggest supplier of paper and board in the world (Molony, 1999 / M

The complex structures of water-soluble wheat arabinoxylans have been mapped along individual molecules, and within populations, using the visualisation of the binding of inactivated enzymes by atomic force microscopy (AFM). It was demonstrated that site-directed mutagenesis (SDM) can be used to produce inactive enzymes as structural probes. For the SDM mutants AFM has been used to compare the binding of different xylanases to arabinoxylans. Xylanase mutant E386A, derived from the Xyn11A enzyme (Neocallimastrix patriciarium), was shown to bind randomly along arabinoxylan molecules. The xylanase binding was also monitored following Aspergillus niger arabinofuranosidase pre-treatment of samples. It was demonstrated that removal of arabinose side chains significantly altered the binding pattern of the inactivated enzyme. Xylanase mutant E246A, derived from the Xyn10A enzyme (Cellvibrio japonicus), was found to show deviations from random binding to the arabinoxylan chains. It is believed that this is due to the effect of a small residual catalytic activity of the enzyme that alters the binding pattern of the probe. Control procedures were developed and assessed to establish that the interactions between the modified xylanases and the arabinoxylans were specific interactions. The experimental data demonstrates the potential for using inactivated enzymes and AFM to probe the structural heterogeneity of individual polysaccharide molecules.

La bioraffinerie, avec l’utilisation de la biomasse végétale comme matière première renouvelable, est un moyen alternatif aux ressources fossiles qui s’amenuisent pour l’obtention de molécules d’intérêt, de biomatériaux ou de biocarburant nécessaires à notre vie quotidienne. Les hémicelluloses constituent 20 à 45% de la biomasse végétale et sont riches en pentoses (D-xylose en particulier) dans le cas des xylanes. Dans le cadre du projet multi-partenaires XYLOCOS, financé par la Région Champagne-Ardenne et le FEDER, l’objectif de cette thèse consiste en l’utilisation d’une xylanase pour mettre au point la préparation par voie enzymatique ou chimio-enzymatique de nouveaux β-xylosides et β-xylobiosides présentant des aglycones de différentes natures et d’évaluer leurs propriétés biologiques. L’étude d’une xylanase GH11 a d’abord été réalisée pour améliorer son activité de transglycosylation en présence de divers accepteurs. Une étude de modélisation in silico a été effectuée et a conduit à cibler la mutation du résidu W126. Les paramètres cinétiques et les propriétés de transglycosylation de la xylanase sauvage et du mutant ont été comparés et ont montré que le mutant a une capacité de transglycosylation améliorée. La synthèse de séries de xylosides et xylobiosides comportant une partie aglycone aromatique par voie enzymatique ou présentant des hétérocycles triazoles diversement fonctionnalisés par voie chimio-enzymatique a été effectuée. L’influence de la nature de la partie aglycone mais également du degré de polymérisation des différents xylosides sur leur capacité à amorcer la biosynthèse de glycosaminoglycanes a été étudiée en présence d’un modèle cellulaire. L’ensemble des xylosides et xylobiosides synthétisés amorce la biosynthèse des GAGs. Les résultats obtenus montrent que les xylosides sont plus efficaces que leurs analogues xylobiosides. Certains xylosides à aglycones hydrophobes représentent de bons candidats pour des applications cosmétiques. / Biorefinery, with the use of plant biomass as a renewable raw material, is an alternative means to fossil resources for obtaining molecules of interest, biomaterials or biofuel. Hemicelluloses represent about 20 to 45% of the plant biomass and are rich in pentoses (D-xylose in particular) in the case of xylans. XYLOCOS is a multi-partner project funded by the Champagne-Ardenne Region and the FEDER. In this context the objective of this work consists in the use of a xylanase to develop the enzymatic or chemo-enzymatic synthesis of new β-xylosides and β-xylobiosides featuring different kinds of aglycone moieties and to assess their biological properties.First, the study of a GH11 xylanase was carried out to improve its transglycosylation activity in the presence of various acceptors. A in silico study was performed and led to target the mutation of W126 residue. Kinetic parameters and transglycosylation properties of the wild and mutant xylanases were compared and showed that the mutant has an improved capacity for transglycosylation.The synthesis of xylosides and xylobiosides bearing an aromatic aglycone part by an enzymatic transformation or functionalized triazole heterocycles by a chemo-enzymatic pathway was performed. The influence of the nature of the aglycone part but also the degree of polymerization of different xylosides was studied through their ability to initiate the biosynthesis of glycosaminoglycans in the presence of a cell model. All the xylosides and xylobiosides prepared act as GAGs primers. The obtained results show that xylosides are more efficient primers than the corresponding xylobiosides. Xylosides carrying hydrophobic aglycones represent good candidates for cosmetic applications.

Dans ce travail, nous avons étudié la contribution que les inhibiteurs de glicosidases ont dans la réponse de défense du blé à Fusarium graminearum. Nous avons démontré que les inhibiteurs de xylanases ont la capacité à la fois de contenir l'activité de dégradation de xylanases sécrétées par l'agent pathogène, et de limiter la possibilité de provoquer nécrose dans les tissus du blé. Nous avons démontré que l’expression de la PvPGIP2 dans la lemme, la paléa, les anthères et le rachis détermine une réduction des symptômes de la fusariose de l’épi, au même niveau de l’expression constitutive de cet inhibiteur. Inversement, l'expression de la PvPGIP2 dans l'endosperme ne détermine pas une réduction des symptômes de la maladie. Cela indique que, lorsque l'agent pathogène a atteint ce tissu, l'activité de polygalacturonases de l'agent pathogène n’est pas indispensable pour la propagation fongique. Enfin, la combinaison des différents inhibiteurs de glicosidases, qui renforcent différentes parties de la paroi cellulaire dans le même génotype, a été efficace pour réduire les symptômes de la fusariose, par rapport aux génotypes qui présentent seulment un type d’inhibiteur. Nous avons démontré cet aspect à travers les plantes qui expriment la PvPGIP2 et le TAXI-III. Au contraire, les génotypes qui expriment la PvPGIP2 et l’AcPMEI n’ont pas montré un effect additif sur la résistance, probablement parce que ils renforcent la même partie de la paroi cellulaire, c’est-à-dire la pectine. / In this work we studied the contribution of glycosidase inhibitors in the defense response of wheat against Fusarium graminearum. We demonstrated that xylanase inhibitors are able to limit both the degrading activity of the xylanases secreted by the pathogen and to limit their ability to induce necrosis in wheat cell suspensions and tissues.We demonstrated that the expression of PvPGIP2 in lemma, palea, anthers and rachis causes a reduction in Fusarium head blight symptoms, at the same level of the constitutive expression of this inhibitor. On the contrary, the expression of PvPGIP2 in the endosperm did not result in a reduction of disease symptoms, suggesting that once the pathogen has reached the endosperm, the activity of polygalacturonases secreted by the pathogen is not essential for the progression of symptoms.The pyramiding of glycosidase inhibitors in the same genotype is effective in reducing FHB symptoms although it depends on the specific combination. Pyramiding of PvPGIP2 and AcPMEI does not enhance further wheat resistance against FHB, possibly because they target the same virulent component secreted by the pathogen, that is PG. Conversely, the pyramiding PvPGIP2 and TAXI-III supports a further improvement of resistance compared to plants carrying only PvPGIP2 or TAXI-III.

Submitted in fulfilment for the requirement of a Degree of Doctor of Technology: Biotechnology, Durban University of Technology, 2010. / The aim of this study was to evaluate T. lanuginosus for the production of hemicellulases, its yield enhancement using mutagenesis and application of a selected xylanase on bagasse pupl to assess the improvement of pulp properties. The objectives were: To determine the localization of hemicellulases in T. lanuginosus strains, To develop high yielding strains of T. lanuginosus through mutagenensis, To investigate the synthesis of xylanase by T. lanuginosus MC134, To optimize the medium components and cultural conitions of T. lanuginosus MC134 strain, To study the influence of agitation and aeration on the production of xylanase by T. lanuginosus MC134 in a fermenter, To evaluate the bleach boosting abilities of T. lanuginosus xylanase on bagasse pulp, To evaluate simultaneous xylanase production and biobleaching potential of T. lanuginosus.

Submitted in complete fulfilment for Masters Degree in Technology: Biotechnology, Durban University of Technology, 2013. / A comparative thermostability analysis of different partially purified xylanases from Rhodothermus marinus, Bacillus halodurans, Thermomyces lanuginosus and Pulpzyme HC was studied using differential scanning fluorometry (DSF), fluorescence spectroscopy and circular dichroism (CD). The R. marinus xylanase was found to have an optimum temperature and pH of 90oC and 6 respectively while the B. halodurans xylanase was optimally active at 70oC and a broad range of alkaline pH of 8 - 10. The commercially available xylanase from T. lanuginosus showed optimal activity at 50oC and pH 7 while the Novozyme xylanase Pulpzyme HC showed optimal activity at 60oC and pH 7. Fluorescence spectroscopy monitored the microenvironment and fluorescence emission of Trp residues. In their native folded state, Trp are generally located in the core of the protein but during unfolding they become exposed. The fluorescence changes as the enzyme undergoes denaturation due to conformational changes and exposure of Trp residues. Differential scanning fluorometry (DSF) monitors thermal unfolding of proteins in the presence of a fluorescent dye such as Spyro Orange. A wide range of buffers were tested for their ability to increase the xylanase stability. T. lanuginosus had the greatest increase in melting temperature with 0.73M Bis Tris pH 6.5 and peaked highest at 78°C. The B. halodurans xylanase exhibited high pH stability (pH 4-10) and exhibited very little change in melting temperature, from 74°C-77°C over the twenty four different conditions. The R. marinus xylanase had no increase in melting temperature showing a maximum melting temperature of 90oC. Circular dichroism (CD) measures unequal absorption of right- and left-handed circularly polarized light by the molecule. The xylanase from R. marinus exhibited the lowest ΔG of 34.71kJ at 90°C as was expected. The B. halodurans xylanase showed a much higher ΔG of -52.71 at its optimum temperature of 70°C when compared with the xylanases from R. marinus and T. lanuginosus. When comparing the three xylanases activities at 70°C, it can be seen that the B. halodurans xylanase exhibited a lower relative activity then both R. marinus and T. lanuginosus xylanases. All three techniques offered different information on the structure and function relationship. Fluorescence spectroscopy, the change in conformation due to fluorescence emission as a result of increased temperature and salt concentrations. DSF, optimal conditions for increased stability and activity at higher temperatures and CD, conformational changes, the fraction of folded protein and change in Gibbs free energy over a range of temperature. / National Research Foundation

Submitted in fulfilment of the requirements for the degree of Master of Engineering, Durban University of Technology, Durban, South Africa, 2015. / Fermentation processes are vital for the production of numerous bioproducts. Fermentation being the mass culture of micro – organisms for the production of some desired product, is an extensive field, with immense prospects for study and improvement. Enzyme production is of significance as these proteins are biological catalysts, finding niches in numerous industries, xylanase for example is utilized in the pulp and paper, animal feed, biofuel and food production processes. During enzyme production, a critical step is biomass separation, whereby the valuable product, the enzyme, is removed from the broth or micro – biological culture before it is denatured. This is typically achieved via centrifugation. The aim of this study was to develop and evaluate a submerged membrane fermenter system with the specific outcome of increasing the rate of production of xylanase, from the thermophilic fungal species Thermomyces lanuginous DSM 5826. Preliminary shake flask experiments were performed to determine the optimal production conditions, followed by partial characterization of the enzyme. A bioreactor was then fabricated to include a flat sheet membrane module, with outlets for permeate and broth withdrawal and inlets for feed and sterile air input. Experiments were conducted to determine the optimal dilution rate for maximum volumetric productivity. Results from the shake flask experiments indicated that the best conditions for xylanase production, yielding xylanase activity of 5118.60 ± 42.76 U.mL-1 was using nutrient medium containing beechwood xylan (1.5 % w/v), yeast extract (1.5 % w/v), potassium dihydrogen phosphate (0.5 % w/v), adjusted to a pH of 6.5 and inoculated with 1.0 mL of spore solution, rotating in a shaking incubator set to 150 rpm at 50 °C. Apart from analysis of the effect of the carbon source on xylanase activity, coarse corn cobs were used in the shake flask experiments as a cost saving initiative. The pH optima was determined to be 6.5 while the temperature optima of the enzyme was 70 °C. SDS PAGE analysis revealed that the molecular weight of the enzyme was between 25 and 35 kDa and qualitative analysis via a zymogram revealed clear zones of hydrolysis on a xylan infused agarose gel. During short run membrane fermenter experiments the percentage increase in enzyme activity between the batch operation (610.58 ± 34.54 U.mL-1) and semi – continuous operation (981.73 ± 55.54 U.mL-1) with beechwood xylan nutrient replenishment was 60.78 %. The maximum volumetric productivity achieved with beechwood supplementation after 192 hours in semi – continuous operation (5.32 ± 0.30 U.mL-1.hr-1) was 2.1 times greater than that of batch operation (2.54 ± 0.14 U.mL-1.hr-1) which equates to an increase of 110.28 % in productivity measured at its peak. The increase in total activity between batch (610 576.92 U) and beechwood xylan medium supplemented semi – continuous mode (1 184 937.50 U) resulted in a 94.07 % increase. During long run experimental periods, the increase in production of xylanase between the batch (873.26 ± 61.78 U.mL-1) and the xylan medium membrane system (1522.41 ± 107.65 U.mL-1) was determined to be 74.34 % while an overall average increase in productivity between the batch and xylan fed membrane system was 43.25%. The total enzyme activity with in membrane mode with beechwood xylan nutrient medium feed was 160 % greater than the batch process offering a 2.6 – fold increase. Experiments where de – ionized water was alternated with beechwood xylan nutrient medium had no significant impact on the productivity or enzyme activity. The optimal dilution rate for maximum volumetric productivity as determined to be 0.0033 hr-1. The results are indicative of the potential viability of such a design, yielding the desired outcome of a membrane integrated system to significantly increase the production of enzymes during fermentation.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Lignocellulose, which is composed of cellulose, hemicellulose and lignin, is the main structural component of plant cell walls. Xylan is the main structural component of hemicellulose. Xylan is a complex heteropolysaccharide and, therefore, requires numerous synergistically acting enzymes for its complete hydrolysis. The focus of this study was on xylanases, which is a main chain cleaving enzyme required for xylan hydrolysis. Xylanases have numerous industrial applications and are commonly used in the biofuels, pulp and paper, food, animal feed and textile industries. Of particular interest is the use of xylanases in the biofuels industry due to the depletion of fossil fuels. A major bottleneck is, however, the low yield and high cost of the enzymatic hydrolysis process. In this study, three different xylanase genes from Aspergillus fumigatus, isolated from a triticale compost heap, were cloned and expressed in Saccharomyces cerevisiae. This yeast is an attractive host for the expression of these heterologous proteins, since A. fumigatus is considered a human pathogen and would not be suited for large-scale enzyme production. The recombinant xylanases obtained in this study were functional after expression in the yeast host and yielded high levels of enzyme activity, ranging from 100 to 300 nkat/mg dry cell weight (DCW). Higher enzyme yields will reduce the overall cost of the enzymatic hydrolysis process, making these enzymes attractive to the biofuels industry. The recombinant xylanases obtained in this study were also free of other cellulases. This characteristic makes these enzymes attractive to the pulp and paper industry as cellulose fibres are required to remain intact. Two of the recombinant xylanases, F10 and F11, were relatively stable at a temperature of 50°C with pH optima at pH 6, while the recombinant xylanase G1 only maintained half of its activity at this temperature and displayed pH optimum at pH 5. No synergistic effect was observed between the recombinant xylanases in this study. Future studies could investigate the synergistic interaction between these recombinant xylanases and other accessory enzymes used for the degradation of xylan, such as the esterases. Xylan hydrolysis levels could increase significantly due to a synergistic effect, which would further reduce the overall cost of the lignocellulose enzyme hydrolysis process. / AFRIKAANSE OPSOMMING: Lignosellulose, saamgestel uit sellulose, hemisellulose en lignien, vorm die hoof strukturele bestanddeel van plantselwande. Xilaan is die hoof strukturele komponent van hemisellulose. Xilaan is ʼn komplekse hetero-polisakkaried en verskeie saamwerkende ensieme vir volledige hidroliese hiervan word benodig. Die fokus van hierdie studie is op xilinases, die hoof kettingbrekende-ensiem vir xilaan hidroliese. Xilinases het verskeie industriële toepassings onder meer in die biobrandstof-, papier en pulp-, voedsel-, dierevoeding- en tekstielindustrieë. Weens die uitputting van fossielbrandstofreserwes word xilinases in die biobrandstof industrie van groot waarde geag. Lae opbrengste en hoë kostes van die ensiemhidroliese proses bly egter ʼn knelpunt. In hierdie studie is drie verskillende xilinase gene vanuit ʼn tritikale komposhoop Aspergillus fumigatus isolaat gekloneer en in Saccharomyces cerevisiae uitgedruk. Gis is ʼn aanloklike gasheer vir die uitdrukking van hierdie heteroloë proteïne aangesien A. fumigatus as menspatogeen nie vir grootskaalse ensiemproduksie geskik is nie. Die rekombinante xilinases verkry in hierdie studie is funksioneel in die gis gasheer uitgedruk en hoë vlakke ensiemaktiwiteit is verkry, van 100 tot 300 nkat/mg droë sel massa (DSM). In die lig van hoër ensiemopbrengste wat die totale koste van die ensiem hidroliese proses verlaag, word die ensieme in hierdie studie aanloklik vir die biobrandstof industrie. Die rekombinante ensieme in hierdie studie verkry is ook vry van ander sellulases, ʼn eienskap wat van waarde is vir die papier en pulp industrie waar die sellulose vesels intak moet bly. Twee van die rekombinante xilinases, F10 en F11, was relatief stabiel by ʼn temperatuur van 50°C met ‘n pH optimum van pH 6, terwyl die rekombinante xilinase G1 slegs die helfte van sy aktiwitieit by hierdie temperatuur kon behou met ʼn pH optimum van pH 5. Geen samewerkende effek kon tussen die drie rekombinante xilinases waargeneem word nie. Toekomstige studies kan die samewerkende effek tussen hierdie rekombinante xilinases en bykomstige ensieme betrokke by xilaanafbraak, soos byvoorbeeld die esterases, ondersoek. Xilaanhidroliese vlakke kan aansienlik as gevolg van hierdie samewerkende effek verhoog, wat die koste van ensiem hidroliese van lignosellulose verder kan verlaag. / Stellenbosch University and the Technology Innovation Agency for financial support

No abstract available. / Thesis (M.Sc.Eng.)-University of KwaZulu-Natal, Durban, 2009.