Cryptococcus laurentii and Cryptococcus flavescens are nonfermenting yeasts forming extracellular polysaccharide capsule. Both species are mainly saprofytic but Cr. laurentii is also known to be an opportunistic pathogen in immunocompromised patients. Cr. flavescens used to be considered a synonym of Cr. laurentii but nowadays it is classified as a separate species that belongs to the phylogenetic group I of the Cr. laurentii group. In the experimental part 28 strains of species Cr. laurentii, Cr. flavescens a Cr. victoriae were biotyped using MALDI-TOF MS. The yeasts were cultivated on three different media (Sabouraud, YPD and potato agar) and three methods were used for the protein extraction. The impact of growth medium composition from which the strains were inoculated on the quality of spectra was studied together with the suitability of individual methods for use on different media. Then the impact of growth medium composition on the quality of acquired spectra was evaluated. Finally, all strains were compared mutually and with the type strain of Cr. laurentii CCY 17 3-2. The composition of the medium cells were inoculated from was found to have little impact on the spectra quality. The same result was determined for the composition of the actual growth medium cells were cultivated on. Crucial for the quality of mass spectrum is the method of cells preparation. Best results were acquired when cultivating cells on YPD agar, washing the cells with ethanol and using mix of sinapinic and ferulic acid as a matrix. Potato agar was found not suitable for cultivating yeasts of the Cryptococcus genus due to significant production of extracellular polysaccharides which complicate the protein isolation process. All strains were compared to Cr. laurentii type strain CCY 17-3-2 and MSP dendrograms were created based on the spectra similarity. In the MSP dendrograms all strains were successfully divided into relevant species on all tested media. Finally sequences of D1/D2 domain of LSU gene were compared and phylogenetic tree was created. This tree was then compared to the MSP dendrograms.

Orientadores: Francisco Maugeri Filho, Gonçalo Amarante Guimarães Pereira / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-25T00:15:25Z (GMT). No. of bitstreams: 1 Andrade_CristianeContePaimde_D.pdf: 8118229 bytes, checksum: e37327c8c0a8a32ecfc89552e46cbd0d (MD5) Previous issue date: 2014 / Resumo: Xilanases são enzimas que hidrolisam as ligações glicosídicas entre as unidades de xilose que compõem a xilana, o principal constituinte hemicelulósico. Devido à grande disponibilidade desse tipo de material na natureza, as xilanases podem ser empregadas em diversos ramos, como nas indústrias têxtil, de alimentos e de rações, na bioconversão de resíduos lignocelulósicos, no clareamento de papel e polpa, e no tratamento de resíduos. Visando descrever novas enzimas para futuras aplicações, este trabalho teve como objetivo purificar e caracterizar a xilanase produzida pelo basidiomiceto Cryptococcus sp. LEB-AY10, uma levedura previamente isolada da Mata Atlântica e selecionada pela produção de enzima termoestável. Para tanto, o micro-organismo foi inicialmente identificado em nível de espécie e depositado como C. flavescens LEB-AY10 (CCT 7725); em sequência, foi estudada a produção da enzima utilizando, como substrato, bagaço de cana-de-açúcar, pré-tratado por explosão a vapor em três diferentes condições, bem como suas respectivas frações solúveis, e suplementados com melaço ou componentes sintéticos. Tendo sido definida a metodologia de tratamento do bagaço, técnicas de planejamento experimental foram utilizadas para estudar a influência das variáveis de cultivo e aumentar a atividade da xilanase produzida. Nas condições otimizadas, obteve-se aumento de 5,6 vezes na atividade em relação aos testes iniciais, atingindo 4,67 U/mL (a 50°C) ou 8,33 U/mL (a 80°C) em 96 h de incubação. Posteriormente, realizou-se a purificação da xilanase em duas etapas cromatográficas (troca iônica e permeação em gel), sendo possível recuperar 45% da atividade inicial. A massa molecular média foi estimada em 48 kDa. A enzima apresentou atividade ótima a 77,5°C e maior estabilidade em pH próximo a 5,3. Neste pH, as meias-vidas desta enzima foram estimadas em 9,2 minutos a 77,5°C e 33,74 h a 67°C. Os parâmetros cinéticos Km e vmax utilizando xilana de bétula (birchwood) como substrato foram 4,13 g/L e 2,32 U/mL, respectivamente. A xilanase purificada apresentou baixas atividades de ?-xilosidase em 4-nitrofenil-?-D-xilopiranosídeo (0,02 U/mg) e de celulase em carboximetilcelulose (0,99 U/mg). Os produtos de hidrólise da xilana de faia (beechwood) pela xilanase de C. flavescens LEB-AY10 foram, principalmente, xilobiose e xilotriose. Para identificação do gene responsável pela produção da xilanase, foram utilizadas três estratégias: amplificação com primers degenerados, identificação de peptídeos por espectrometria de massas e sequenciamento do genoma da levedura. Um único gene (1265 pb) denominado Xyn10Cf foi identificado e o correspondente cDNA (1035 pb) foi clonado em Escherichia coli DH10B. O gene é interrompido por 6 íntrons, codifica um peptídeo sinal composto por 13 aminoácidos e a proteína madura é formada por 331 aminoácidos, tendo sua massa molecular estimada em 37,1 kDa (podendo conter cerca de 11 kDa de glicosilação). Com base na sequência de nucleotídeos e na caracterização da enzima purificada, a proteína foi classificada como membro da família GH10. Por fim, para comprovar a funcionalidade do gene identificado Xyn10Cf foi realizada a expressão na linhagem de Pichia pastoris GS115 sob o controle dos promotores álcool oxidase 1 (AOX1) ou gliceraldeído-3-fosfato desidrogenase (GAPDH), atingindo 5,66 U/mL e 7,67 U/mL, respectivamente, para 84 h de indução em frascos agitados / Abstract: Xylanases are enzymes that hydrolyze the glycosidic bonds between the xylose units of xylan, the main hemicellulosic compound. Due to the high availability in nature of this kind of material, the xylanases may be used in a wide range of industrial plants, including: textile, food and feed industries, bioconversion of lignocellulosic wastes, biobleaching of paper and pulp, and waste treatment. In order to describe novel enzymes for future applications, the goal of this work is to purify and characterize the xylanase produced by the basidiomycete Cryptococcus sp. LEB-AY10, a yeast previously isolated in the Atlantic Forest and selected by the production of a thermostable enzyme. First, the microorganism was identified at the species level and deposited as C. flavescens LEB-AY10 (CCT 7725); then, enzyme production was studied using the substrate sugarcane bagasse, pretreated by steam explosion in three different conditions, as well their corresponding soluble fractions, and supplemented with molasses or synthetic compounds. After selection of the treatment, experimental design techniques were used to assess the influence of cultivation variables and to increase the activity of produced xylanase. At optimized conditions, it was possible to increase activity by 5.6 times from initial assays, reaching 4.67 U/mL (at 50°C) or 8.33 U/mL (at 80°C) after 96 h of incubation. Later, the purification of the xylanase was performed in two chromatographic steps (ion exchange and gel permeation), in which it was possible to recover 45% of initial activity. The average molecular weight was estimated at 48 kDa. The enzyme showed optimal activity at 77.5°C and it was most stable at pH values near 5.3. At this pH, the half-lives of this enzyme were 9.2 min at 77.5°C and 33.74 h at 67°C. The kinetic parameters Km and vmax for Birchwood xylan were 4.13 g/L and 2.32 U/mL, respectively. The purified xylanase showed low activity of ?-xylosidase on 4-nitrophenyl-?-D-xilopiranosídeo (0.02 U/mg) and also showed some cellulase activity on carboxymethylcellulose (0.99 U/mg). The hydrolysis products of Beechwood xylan by the xylanase from C. flavescens LEB-AY10 were mainly xylobiose and xylotriose. For identification of the gene responsible for xylanase production, three strategies were used: amplification of degenerated primers, identification of peptides by mass spectrometry and yeast genome sequencing. A single gene (1265 pb) named Xyn10Cf was identified and the corresponding cDNA (1035 pb) was cloned into Escherichia coli DH10B. The gene is interrupted by 6 introns, it codifies a signal peptide of 13 amino acids and the mature protein is composed by 331 amino acids, with an estimated molecular mass of 37.1 kDa (it may contain around 11 kDa of glycosilation). Based on nucleotide sequencing and on characterization of the purified enzyme, the protein was classified as a member of GH10 family. Finally, to prove the functionality of the identified gene Xyn10Cf, the expression in Pichia pastoris GS115 strain was performed under the control of alcohol oxidase 1 (AOX1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoters, reaching 5.66 U/mL and 7.67 U/mL, respectively, after 84 h of incubation in shaker flasks / Doutorado / Engenharia de Alimentos / Doutora em Engenharia de Alimentos

No description available.