Orientador: Rubens Monti / Banca: Sandra Regina Pombeiro Sponchiado / Banca: Eleonora Cano Carmona / Resumo: Neste trabalho foi utilizada a metodologia de superfície de resposta, por meio de delineamento composto central rotacional para investigar as melhores condições de produção de xilanase pelo fungo filamentoso Aspergillus niger. Este micro-organismo é considerado um bom produtor de enzimas xilanases, sendo que estas enzimas têm a capacidade de hidrolisar xilana em xilooligossacarídeos e xilose. Produtos assim obtidos estão sendo cada vez mais utilizados em rações animais para melhoria da flora intestinal; para a produção de xilitol e também para a produção de álcool de segunda geração. A análise estatística dos resultados obtidos neste trabalho mostrou que as melhores condições de produção da enzima extracelular foram: pH 5,0, temperatura de 37 ºC, agitação de 80 rpm, e concentração de fonte de carbono de 2 % (p/v). Após a determinação das condições ideais, o extrato foi clarificado por filtração em caulim, e as proteínas assim obtidas foram precipitadas com acetona ocorrendo uma melhora sensível na atividade específica. Após filtração em Sephadex G-75 foi mostrada a presença de atividade xilanolítica em dois picos, e as frações referentes ao segundo pico foram reunidas e submetidas à coluna de troca iônica DEAE-Trisacryl, na qual se constatou uma fração sendo eluída com 0,06 mol/L de NaCl, contendo atividade de xilanase. A SDS-PAGE da fração majoritária revelou uma única banda protéica com massa molar aparente de 34 kDa. A cromatografia em sílica gel P60 revelou que os produtos de hidrólise foram constituídos de xilooligossacarídeos, após 120 min de hidrólise / Abstract: In the present work, response surface methodology was utilized, through appliance of rotational central composite design to investigate the best conditions of production of xylanase by the filamentous fungus Aspergillus niger. This microorganism is considered a good producer of xylanase enzyme, which has the ability to hydrolyze hemicelluloses in xylooligosaccharides and xylose. Products obtained this way are being increasingly utilized in animal feeding to improve the intestinal flora, to produce xylitol and also to produce second generation ethanol. The statistical analysis of the obtained results showed that the best conditions for the extracellular enzyme production were: pH 5.0, 37 ºC, 80 rpm shaking, and 2 % (w/v) carbon source concentration. After the determination of the ideal production conditions, the enzyme extract was clarified through filtration in kaolin, and the protein obtained were precipitated with acetone, with a sensitive increase in the specific activity. After molecular exclusion chromatography in Sephadex G-75 the presence of xylanolitic activity was shown in two peaks, and the fractions related to the second peak were collected and submitted to DEAE-Trisacryl ion exchange column, in which were observed fractions showing xylanase activity that was eluted with 0.06 mol/L NaCl. SDS-PAGE of the majority fraction revealed only one proteic band with apparent molar mass of 34 kDa. P60 silica gel chromatography revealed that the product of hydrolysis was constituted of small xylooligosaccharides released after 120 min of hydrolysis / Mestre

Orientador: Rubens Monti / Banca: Daniela Alonso Bocchini Martins / Banca: Maristela de Freitas Sanches Perez / Banca: Eleonora Cano Carmona / Banca: Marcos Filice / Resumo: As xilanases são glicosidases que catalisam a hidrólise das ligações 1,4-β-xilosídicas da xilana e que possuem potencial biotecnológico em vários processos industriais, como na clarificação de sucos e vinhos, na fabricação de pães, na filtração da cerveja e no tratamento das polpas celulósicas. Recentemente, recebem atenção pela produção dos xilooligossacarídeos como ingredientes prebióticos. Embora possuam diversas vantagens sobre os métodos químicos, as enzimas são, geralmente, limitadas para uso industrial. A imobilização em suportes sólidos melhora a estabilidade dos biocatalisadores e o controle operacional, promovendo a recuperação do produto sem a contaminação pela enzima. Assim, os objetivos deste trabalho foram: produzir e caracterizar a xilanase de Aspergillus niger; imobilizar covalentemente, em suportes sólidos, a xilanase de A. niger e quatro outras, provenientes de diferentes fontes (Aspergillus versicolor, Trichoderma longibrachiatum, Thermomyces lanuginosus e Streptomyces halstedii); caracterizar os derivados obtidos; analisar o produto de hidrólise da xilana pelos derivados. A xilanase de A. niger mostrou ótima estabilidade até 55°C, com meia-vida de 15 minutos a 60°C, e sua produção foi induzida por pequenas concentrações de xilose. O fungo secretou pelo menos duas isoformas com atividade xilanolítica. A xilanase I foi purificada em uma única etapa, por adsorção em suporte ativado com quelatos, assim como a enzima de S. halstedii, contendo cauda de histidina. A xilanase de T. longibrachiatum (comercial) foi parcialmente purificada com suportes iônicos, e as de T. lanuginosus (comercial) e A. versicolor já apresentavam alto grau de pureza. As enzimas foram imobilizadas em agarose ativada com diferentes grupos reativos, com fatores de estabilização entre 12 (T. lanuginosus) e 600... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Xylanases are glycosidases that catalyze the hydrolytic cleavage of β-1,4-linked polymers of D-xylose and they have biotechnological potential in various industrial processes such as in the clarification of juices and wines, in the manufacturing of breads, in beer filtration and in the treatment of cellulose pulps. Recently, attention is given to the production of xylo-oligosaccharides as prebiotic ingredients. While enzymes have several advantages over chemical methods, they are generally limited for industrial use. Immobilization on solid supports improves the stability of the biocatalyst and the operational control, and promotes the easy recovery of the product without contamination by the enzyme. The objectives of this study were: to produce and characterize xylanases from Aspergillus niger; to immobilize the xylanase of Aspergillus niger and four others from different sources (Aspergillus versicolor, Trichoderma longibrachiatum, Thermomyces lanuginosus and Streptomyces halstedii) covalently on solid supports; to characterize the derivatives obtained; to analyze the products profile of xylan hydrolysis by the derivatives. The xylanase of A. niger showed excellent stability up to 55°C, with a half-life of 15 minutes at 60°C, and its production was induced by low concentrations of xylose. The fungus produced at least two isoforms with xylanolytic activity. Xylanase I was purified in one step by adsorption on support activated with chelates, as well as the histidine-tagged enzyme of S. halstedii. The xylanase of T. longibrachiatum (commercial) was partially purified with ionic supports, and those from T. lanuginosus (commercial) and A. versicolor already showed high degree of purity. The xylanases were then immobilized on agarose activated with different reactive groups, and presented stabilization factors between 12 (T. lanuginosus)... (Complete abstract click electronic access below) / Doutor

Submitted in fulfillment for the requirement of a Degree of Master in Biotechnology, Durban University of Technology, 2017. / Microbial xylanases have attracted considerable research interest because of their various applications in biotechnology including the biobleaching of kraft pulp, to increase the nutritional value of foods and animal feed as well as for their potential use in the production of ethanol and methane. In the paper and pulp industry, the bleaching process involves the use of toxic chemicals and in the interim produces harmful gases that have a negative impact on the environment. The application of enzymes for this process will potentially reduce the environmental pollution by this industry. In addition, using an enzyme that is thermostable and alkali tolerant means that they will remain active under the required processing conditions. The xylanase gene, xynA derived from Thermomyces lanuginosus DSM 5826, was previously evolved to produce a number of xylanase variants, which were further enhanced for increased thermostability and alkalinity. In this study, these variants were cloned in Pichia pastoris using the pBGP1 vector to achieve extracellular production of the recombinant proteins. The xylanase genes were isolated using PCR. Both vector and DNA inserts were linearized with restriction enzymes EcoRI and XbaI and ligated. Electroporation was employed to transform the yeast with the recombinant plasmids. This was followed by the expression of the enzymes in P. pastoris grown in yeast peptone glucose (YPD) medium. Enzyme activity was thereafter assessed and the yeast was found to produce 164, 78, 96 and 142 IU/ml of S325, S340, G41 and G53 xylanase respectively, higher levels than bacterial hosts. The enzymes were then characterized and it was established that the optimum temperatures and pH for maximum xylanase activity were, 60°C, pH 6 for S325; 40°C, pH 5 for S340; 60°C, pH 6 for G41 and 60°C, pH 7 for G53. i The pH and temperature stabilities of the respective enzymes were investigated, the S325 variant was exceptionally stable at a pH between 5 and 7 and temperature range of 40-80°C and retained a minimum of 40% of activity at higher pH and temperature after an incubation period of 90 min. The S340 variant was the least thermostable and alkali stable from all four variants, it however retained 40% of activity when subjected to conditions of pH 9, 80°C after 90 min. The G41 and G53 were highly stable under the pH and temperature conditions that they were subjected to. Thus being suitable for potential application in the pulp and paper industry. The enzymes were able to retain 80% of activity at pH 9, 80°C after 120 min. P. pastoris has been proven to be a more suitable protein expression vector than E. coli for a number of reasons, including; the ability to perform complex post-translational modifications and grow to high densities in minimal media resulting in the production of a high yield of heterologous proteins. / M

>Magister Scientiae - MSc / Lignocellulosic biomass, a waste component of the agricultural industry, is a promising source for use in bioethanol production. Due to a complex structure, the synergistic action of lignocellulosic enzymes is required to achieve complete digestion to fermentable sugars. This study aimed to isolate, identify and characterise novel lignocellulase producing bacteria from thermophilic straw-based compost (71°C). Colonies with different morphological characteristics were isolated and screened for lignocellulosic activity. A facultative aerobic isolate RZ1 showed xylanase, cellulase and lipase/esterase activity. In addition to these activities, it was also able to produce proteases, catalases, amylases and gelatinases. RZ1 cells were motile, rod-shaped, Gram positive and endospore forming. The growth temperature of isolate RZ1 ranged from 25-55°C with optimal growth at 37°C. The 16S rRNA gene sequence was 99% identical to that of Bacillus subtilis strain MSB10. Based on the biochemical and physiological characteristics and 16S rRNA gene sequence, isolate RZ1 is considered a member of the species B. subtilis. A small insert genomic library with an average insert size of 5 kb was constructed and screened for lignocellulosic activity. An E.coli plasmid clone harbouring a 4.9 kb gDNA fragment tested positive for xylanase activity. The xyl R gene was identified with the aid of transposon mutagenesis and the deduced amino acid sequence showed 99% similarity to an endo-1-4-β-xylanase from B. pumilus. High levels of xylanases were produced when isolate RZ1 was cultured (37°C) with beechwood xylan as a carbon source. On the other hand, the production of xylanases was inhibited in the presence of xylose. Marked xylanase activity was measured in the presence of sugarcane bagasse, a natural lignocellulosic substrate. While active at 50°C, higher xylanase activity was detected at 37°C. Isolate RZ1 also produced accessory enzymes such as β-xylosidases and α-L-arabinofuranosidases, able to hydrolyse hemicellulose.

Xylan is the major component of hemicellulose and its degradation can be achieved through the hydrolytic action of microbial xylanases. Xylanases have an array of applications one being bioethanol production. The lack of thermophilic xylanases has prompted the search for new enzymes with increased thermostability. Previous work on the crude enzyme of Phialophora alba has demonstrated optimal activity (39 U/μg) at a pH of 4 and two temperature optima of 50°C and 90°C. These desirable properties highlighted the need for further research on the purified enzyme. In the present study P. alba was identified as a thermophilc Ascomycete that forms conidia and chlamydospores during the asexual and sexual stages of its life cycle, respectively. The various isozymes present in the crude enzyme extract were subsequently detected by zymogram analysis. Up to six xylanase isozymes ranging from 90-210 kDa in size were detected. The crude enzyme was subsequently purified by precipitation and ion exchange chromatography (IEX). Protein precipitation methods, desalting methods, IEX resins, elution buffers and NaCl gradients were optimized. The 31-70% ammonium sulphate precipitate had the highest levels of xylanase activity. Separation of proteins with the anion exchanger, HiTrap Q sepharose fast flow column and a linear gradient of 0-2.5 M NaCl in phosphate buffer (50 mM, pH 7) yielded a partially pure xylanase isozyme with molecular weight of 210 kDa. A final yield of 1.4% and purification fold 10.6 was obtained after ion exchange chromatography. The specific activity of the xylanase was 21 IU/μg. At optimum pH (pH 4) and temperature (50°C) a combined xylanase activity of 32 IU.ml⁻¹ was detected. The partially pure xylanase was stable from pH 4-6 with 86% of xylanase activity retained for 90 minutes. Thermostability was observed from 40-70°C with 95% of activity retained for 90 minutes at optimum temperature. The ability of the partially pure xylanase and crude enzyme to hydrolyze untreated and pretreated (alkali and temperature/pressure) sugarcane bagasse was tested at a constant enzyme loading rate of 15 IU/g. Overall, maximum hydrolysis was achieved with the alkali pretreatment and saccharification with the crude enzyme: approximately, 2.4 g/ml of reducing sugars were liberated over a 48 hours. The partially pure xylanase liberated a maximum amount of 2.3 g/ml reducing sugars after 48 hours. The results obtained highlight the desirable characteristics of the partially pure enzyme and its applicability to bioethanol production. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2013.

Thesis (Ph. D.)--University of Hawaii at Manoa, 2005. / Includes bibliographical references (leaves 120-148). / Also available by subscription via World Wide Web / xv, 148 leaves, bound col. ill., col. charts 29 cm

Thesis (Ph. D.)--University of Adelaide, Dept. of Plant Science, 1997. / Includes bibliographical references (leaves 127-166).

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

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. / Submitted by Ana Guimarães Pereira (agpereir@ucs.br) on 2015-09-22T17:54:12Z No. of bitstreams: 1 Dissertacao Denise Zampieri.pdf: 18671785 bytes, checksum: 0c85c4e913bd6377f21bfe2a1fe9e8ff (MD5) / Made available in DSpace on 2015-09-22T17:54:12Z (GMT). No. of bitstreams: 1 Dissertacao Denise Zampieri.pdf: 18671785 bytes, checksum: 0c85c4e913bd6377f21bfe2a1fe9e8ff (MD5) / 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.