Orientador: Helia Harumi Sato / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-01T14:14:34Z (GMT). No. of bitstreams: 1 Ferro_LilianAparecida_D.pdf: 39343182 bytes, checksum: 85dd4ff7fee79e9940cf96d45fae535d (MD5) Previous issue date: 2002 / Resumo: Este trabalho objetivou o isolamento de microrganismos capazes de produzir enzimas que lisam a parede celular de leveduras, a produção, purificação e caracterização de 13-1,3-glucanase lítica. As bactérias líticas foram isoladas de Iodo da usina de açúcar e álcool Santa Helena, Piracicaba, SP. As bactérias líticas isoladas aderiram-se às células viáveis de Saccharomyces cerevisiae 701 e lisaram-nas. A linhagem YLM-B191-1, selecionada para o estudo, foi identificada através das características bioquímicas e fisiológicas como Cellulomonas cellulans. Para a produção da 13-1,3-glucanase lítica, a linhagem de C. cellulans YLM-B191-1 foi cultivada em meio composto de 15 9 de levedura seca; 2,0 9 de (NH4hSO4; 13,6 9 de KH2PO4; 4,2 9 de KOH; 0,2 9 de MgSO47H2O; 0,001 9 de Fe2(SO4h 6H2O; 1 mg de biotina e 1 mg de tiamina por litro. A 13-1,3-glucanase foi purificada do sobrenadante do meio de cultura através de ultrafiltração e cromatografia em coluna de CM-Sepharose CL-6B. A enzima purificada apresentou atividade ótima a 55°C e na faixa de pH entre 4,5 a 6,5. A 13-1,3- glucanase purificada apresentou estabilidade na faixa de pH 5,5 a 6,5 e foi inativada em temperaturas superiores a 55°C. A massa molecular da 13-1,3- glucanase purificada foi estimada em 17,1 kDa através de SDS-PAGE. A 13-1,3- glucanase purificada hidrolisou as ligações 13-1,3-glicosídicas da laminarina atuando como uma endoenzima. Através de microscopia eletrônica de varredura, observou- se que as enzimas líticas da linhagem de C. cellulans YLM-B191-1 foram capazes de alterar a superfície celular de leveduras / Abstract: The objective of this research was to isolate microorganisms which produced yeast cell walllytic enzymes and to study the production, purification and characterization of a Iytic ~-1,3-glucanase. The yeast-Iytic bacterium was isolated from the sludge of the Santa Helena sugar and alcohol factory in Piracicaba, SP. The isolated yeast-Iytic bacterium adhered to viable cells of Saccharomyces cerevisiae701 and Iysed them.The yeast-IyticbacteriumYLM-8191-1, selected for this study, was identified as Cellulomonas cellulans, from its biochemical and physiological characteristics.The strain C. cellulansYLM 8191-1 was cultivated in a medium containing (per liter) 2.0 9 of (NH4hSO4; 13.6 9 of KH2PO4;4.2 9 of KOH; 0.2 9 of MgSO47H2O; 0.001 9 of Fe2(SO4h6H2Oand 1 mg each of biotin and thiamin being supplemented with 15 9 of dried yeast as the carbon source for the production of ~-1,3-glucanase. The ~1 ,3-glucanase was purified from the culture fluid of C. cellulans YLM-8191-1 by ultrafiltration and CM-Sepharose CL-68 column chromatography. The purified enzyme showed greatest activity at 55°C and between pH 4.5 - 6.5. The purified ~-1,3-glucanase was stable in the range from pH 5.5 to 6.5 and was inactivated by heating at temperatures above 55°C. The molecular weight of purified ~-1,3-glucanasewas estimated at about 17.1 kDa by SDS-PAGE. The ~-1,3-glucanase hydrolyses the ~-1,3-glucosidic linkages of the laminarin acting as an endoenzyme. Scanning electron microscopy showed that Iytic enzymes from C. cellulans YLM-8191-1 were able to modify the cellular surface of yeast / Doutorado / Doutor em Ciência de Alimentos

The Bacillus anthracis spore is highly resistant to environmental stresses, but cannot cause anthrax until it successfully germinates. An essential step of germination, degradation of the cortex peptidoglycan layer, is carried out by germination-specific lytic enzymes (GSLEs). While the GSLEs of several other Bacillus species have been investigated, they have not been characterized in the pathogen B. anthracis. In this work three GSLEs, CwlJ1, CwlJ2, and SleB are identified in B. anthracis and are investigated in order to better understand their functions. Genetic manipulation of cwlJ1, cwlJ2, and sleB was fundamental to this work. First, reporter gene fusions revealed that all three are expressed during spore formation and that CwlJ1 is likely the most abundant GSLE in the spore. Second, gene deletions eliminating each GSLE enabled the observation of mutant phenotypes during spore cortex degradation. CwlJ1 and SleB were identified as the most critical GSLEs for successful germination. High-performance liquid chromatography and mass spectroscopy revealed that SleB is required for lytic transglycosylase activity, but CwlJ1's mode of action was unclear. Multiple mutations of all of the GSLEs revealed that CwlJ2 is the least active GSLE, but that it participates in germination in response to Ca-Dipicolinic acid; a role it shares with the more dominant CwlJ1. Purification of the CwlJ1 and SleB proteins permitted in vitro assays of enzymatic activity as measured by changes in substrate optical density, solubility, and product formation. While CwlJ1 was recalcitrant to these methods, it was observed to cause cortex hydrolysis independently. SleB was more amenable and it was discovered to contain a peptidoglycan-binding domain that is primarily responsible for substrate binding, and a lytic transglycosylase domain that facilitates cortex-specific hydrolysis by recognizing muramic-δ-lactam. Future research will include determining the structure of SleB through x-ray crystallography and the identification of CwlJ1 activity by refining the protein purification method. The results of this and future research into CwlJ1, CwlJ2, and SleB may lead to a means to initiate spore germination prior to host infection. This will greatly ease spore decontamination measures, lower risk of infection, and discourage the use of B. anthracis spores as a biological weapon. / Ph. D.