Desenvolvimento de um bioprocesso para a produção, caracterização e recuperação da Fitase de Schizophyllum Commune obtida por fermentação em estado sólido

Orientadora: Profa. Dra. Michele Rigon SpierCo-orientadores: Prof. Dr. Carlos Ricardo Soccol, Profa. Dra. Luciana P.S. VandenbergheDissertação (mestrado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduaçao em Processos Biotecnológicos. Defesa: Curitiba, 29/04/2011Bibliografia: fls. 91-107Área de concentração:Agroindústria e BiocombustíveisResumo: As fitases hidrolisam o acido fitico em inositol e fosfatos, os quais se encontram armazenados em graos, sementes e legumes. Fitases podem ser produzidas por processos de fermentacao submersa (FSm) ou fermentacao no estado solido (FES). A FES e a mais vantajosa, pois pode utilizar residuos agroindustriais como substrato/suporte, os quais apresentam baixo custo e possuem elevado rendimento de producao final. As fitases apresentam aplicacoes em racao animal e produtos para consumo humano. Estas melhoram a absorcao do fosforo, de outros nutrientes no organismo e tambem reduzem a quantidade de fosforo eliminado nos excrementos dos animais resultando em beneficios para o meio ambiente. O presente trabalho teve como objetivo otimizar a producao da enzima fitase de S. commune por FES utilizando o farelo de trigo como substrato/suporte, caracterizar, recuperar a fitase produzida, estudar a extracao liquido-liquido da fitase alem de estudar a sua estabilidade durante o armazenamento. O basidiomiceto Schizophyllum commune foi selecionado como um produtor de enzima fitase utilizando o farelo de trigo como substrato/suporte para a producao de fitase. Para a otimizacao da producao de fitase foi realizado um planejamento experimental fatorial fracionado Box-Behncken design 35 utilizando cinco variaveis independentes (temperatura, concentracao de sacarose, concentracao de extrato de levedura, pH e taxa de inoculo) em tres niveis (+1, 0, -1) e cinco pontos centrais totalizando 37 experimentos. A producao maxima de fitase (113,76 U/gbs) foi obtida com o meio suplementado com sacarose 5%, pH 7, taxa de inoculo 7,5% e temperatura de fermentacao de 33 ‹C. Esse resultado aumentou 285% a producao de fitase em 72 horas de fermentacao. A fitase apresentou uma atividade otima em pH 5 e temperatura de 50 ‹C, Km e Vmax de 0,16 mM e 1,85 ƒÊmol/mL.min respectivamente. A fitase foi ativada na presenca de K+, Ca2+, Mg2+, Mn2+, Zn2+, Cu2+, Fe2+, Fe3+, Co2+, Ni2+, Na+ acetato e citrato e completamente inibida por molibdato de amonio. A melhor condicao de armazenamento para a manutencao da estabilidade da enzima sob refrigeracao (4 ‹C) com 22% de atividade relativa em 125 dias. Estudos preliminares com estabilizantes no extrato bruto enzimatico foram realizados apontando o aditivo A (0,25% p/v) como o melhor agente estabilizante o qual mantem 109% de atividade relativa durante 90 dias de armazenamento a temperatura ambiente de 25 ‹C. A extracao liquido-liquido da enzima utilizando as condicoes de concentracao de citrato 14% (m/m), massa molar de PEG 1500, concentracao do PEG 22% (m/m) e pH 7 tambem apresentou um resultado satisfatorio, com recuperacao de 367 %, fator de purificacao de 5,43 e coeficiente de particao de 2,63.Abstract: Phytases hydrolyze phytic acid to inositol and phosphates, which are stored in grains, seeds and vegetables. Phytases can be produced by submerged fermentation (SmF) and solid-state fermentation which is most used and commercially advantageous. It can use agroindustrial residues as substrate/support, which reduces the cost of the bioprocess and the final price of the enzyme. Phytases have applications in feed and products for human consumption. Phytases improve phosphorus absorption and others nutrients and also reduce the amount of phosphorus eliminated in the animals excrements resulting in benefits to the environment. This study aimed to optimize the phytase production of S. commune by SSF, characterize, recovery the produced phytase, study the liquid-liquid extraction and the stability during storage. The basidiomycete Schizophyllum commune was selected as a major of phytase producer using wheat bran as substrate/support for the phytase production. The optimization of phytase production was carried out by a full 35 fractional factorial Box-Behnken experimental designs using five independent variables (temperature, sucrose concentration, yeast extract concentration, pH and inoculum rate) at two levels (+1, 0, -1) and five central points totalizing 37 experiments. The maximal level of phytase (113.76 U/gds) was obtained in a medium containing sucrose 5%, pH 7.0, inoculum rate 7.5% at 33ºC during 72 hours. This result increased 285% the phytase production in 72 hours fermentation. The enzyme had an optimum pH 5 and 50°C, Km and Vmax of 0.16 mM and 1.85 mmol / mL min, respectively. The enzyme was activated in the presence of K+, Ca2 +, Mg2 +, Mn2 +, Zn2 +, Cu2 +, Fe2 +, Fe3 +, Co2 +, Ni2+, Na+, acetate and citrate and completely inhibited by ammonium molybdate. The best storage condition for maintaining the enzyme stability was at 4 ° C, with 22% relative activity in 125 days. Preliminary studies with stabilizers agents in crude enzymatic extract were performed, resulting in additive A (0.25% w/v) as the best stabilizing agent with 109% relative activity during 90 days of storage at room temperature 25°C. The liquid liquid extraction of ezyme using citrate 14% (m/m), PEG 1500, PEG concentration of 22% (w/w) and pH 7 also had satisfactory results, with a partition coefficient of 2.63, yield 367% and purification factor of 5.43

Desenvolvimento de um bioprocesso para a produção, recuperação e formulação de fitase termoestável de Ganoderna sp. MR-56 obtida por cultivo submerso

Orientadora: Prof(a). Dr(a). Michele Rigon SpierCoorientadores: Prof. Dr. Carlos Ricardo Soccol, Prof(a). Dr(a). Luciana P.S. VandenbergheTese (doutorado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Defesa : Curitiba, 12/02/2015Inclui referênciasResumo: A presente tese teve como objetivo a produção de fitase termoestável de Ganoderma sp. MR-56 empregando o extrato de farelo de trigo, um subproduto agroindustrial, como substrato para o cultivo submerso. As fitases são enzimas comumente utilizadas em processamento de ração animal e alimentos para catalisar a degradação do ácido fítico em inositol e fosfatos. O ácido fítico é considerado um fator anti-nutricional presente nos vegetais, pois diminui a biodisponibilidade de alguns nutrientes em aves e suínos. Dessa forma, a aplicação da fitase reduz a adição de fosfato inorgânico nas rações, consequentemente, um benefício econômico e a diminuição da excreção desse mineral no meio ambiente. Atualmente as fitases produzidas industrialmente têm origem microbiana e são obtidas por processos fermentativos. Porém, a produção de fitases de macromicetos ainda é limitada e por cultivos submersos nunca reportados na literatura até o presente estudo. A tese teve como objetivo identificar o micro-organismo produtor de fitase e otimizar a produção da enzima. Além disso, a recuperação, caracterização, formulação e o estudo da estabilidade da enzima também foram realizados. O Ganoderma sp. foi identificado por métodos morfológico e molecular. A otimização da produção de fitase foi constituída por 2 etapas: a primeira consistiu em um delineamento experimental do tipo DCCR (Delineamento Composto Central Rotacional) para estudar a suplementação do meio de cultivo com melaço de soja, extrato de levedura e cloreto de cálcio. A segunda etapa da otimização foi constituída de um DCCR e os fatores estudados foram temperatura de cultivo, pH inicial e taxa de inoculação. O meio de cultivo otimizado continha extrato de levedura 8% m/v, temperatura de 30°C, pH 6,0 e taxa de inoculação de 3% v/v e a produção máxima de fitase foi de 14,5 U mL-1. Nos estudos de cultivo submerso em diferentes tipos de biorreatores, o frasco tipo Dreschel com 2 vvm resultou na melhor produção da enzima de 20,94 U mL-1, porém o STR alcaçou uma melhor produtividade de fitase de 0,14 U-1mL-1h-1. A concentração por ultrafiltração em dispositivos de centrifugação em tubos com poro de 30 kDa apresentou maior concentração da enzima em 36,75 vezes. As estratégias de purificação de cromatografia de troca iônica, filtração em gel S-100 e S-400 não foram eficientes para a separação da fitase. Por outro lado, o zimograma indicou bandas com atividade de fitase as quais apresentaram elevada massa molecular. A fitase foi caracterizada e apresentou pH ótimo entre 4,5 e 5,0, e adequada termoestabilidade à 80 e 90ºC/30 min com 100 e 94,24% de atividade residual, respectivamente. A fitase foi ativada por Mn2+, Ca2+, Na+, Co2+, Fe2+, EDTA e fosfato inorgânico na concentração de 1 mM. Após seriados estudos de formulação líquida com emprego de aditivos, verificou-se que a presença do antimicrobiano A1 0,21% e antioxidante O2 0,0021% foram importantes para a manutenção da estabilidade da enzima durante o armazenamento (103,5% de atividade relativa) em 15 dias em condições aceleradas (40ºC). Os estudos de armazenamento à temperatura ambiente da fitase em pó resultaram que o polímero E2 apresentou 79% de atividade residual após 90 dias de estudo. A fitase produzida e formulada apresentou um potencial para possível aplicação em ração animal e produtos para consumo humano com o intuito de diminuir o fitato, bem como melhorar a absorção de alguns nutrientes. Palavras-chave: fitase, Ganoderma sp. MR-56, otimização, concentração, formulação, aditivos e spray-drying.Abstract: The aim of this thesis was the production of a thermostable phytase from Ganoderma sp. MR-56 by submerged culture, using wheat bran extract as agroindustrial by-product. Phytases are enzymes employed in processing feed and food to catalyze the degradation of phytic acid to inositol and phosphates. The phytic acid is considered an antinutritional factor present in vegetables and decreases the bioavailability of some nutrients in poultry and pigs. The phytase application reduces the inorganic phosphate addition on feed, therefore, an economic benefit and diminished excretion of this mineral into the environment. Currently, phytases from microbial source have been obtained by industrial fermentation processes. However, phytases production by macromycetes is still limited and by submerged culture production had never been reported before in the literature. This thesis aims to identify the microorganism producing phytase and optimize enzyme production by submerged culture. In addition, recovery, characterization, formulation and stability studies of phytase were performed. The Ganoderma sp. was identified by morphological and molecular methods. The optimization of phytase bioprocess was composed by two steps: the first consisted of an experimental design CCRD two levels and three factors to study the submerged culture medium supplementation composed of soybean molasses, yeast extract and calcium chloride. The second optimization consisted of CCRD and the factors studied were culture temperature, initial pH and inoculum rate. The optimized medium contained 8% w/v yeast extract, temperature of 30°C, pH 6.0 and 3% v/v inoculum rate and the maximum phytase production reached 14.5 U mL-1. Studies of different bioreactors for phytase synthesis, the Dreschel bottle type with 2 vvm was the best for enzyme production (20.94 U mL-1), however in STR reached the best phytase productivity of 0,14 U-1mL- 1h-1. The ultrafiltration tubes using 30 kDa fraction retained was 36.75-fold of concentrated enzyme. Strategies purification, of ion exchange chromatography, gel filtration S-100 and S-400 were not effective for the phytase separation. Phytase zymogram suggested activity bands with estimated high molecular weight. The enzyme was characterized and had optimum pH of 5.0 and a thermo stability phytase at 80 to 90ºC for 30 min showed 100 and 94.24% of residual activity, respectively. The enzyme was activated by Mn2+, Ca2+, Na+, Co2+ and Fe2 +, EDTA and inorganic phosphate at concentrations of 1 mM. After series of studies for phytase liquid formulation additives, it was found that the antimicrobial 0.21% A1 and antioxidant 0.0021% O2 were important to maintain enzyme stability during storage 103.5% phytase residual activity on 15 days at 40°C. The formulation powder studies allowed select the polymer E2 for phytase encapsulation with 79% phytase activity relative at room temperature after 90 days of storage. The produced and formulated phytase presented a potential application for feed and processed food in order to reduce the phytate as well as improving some nutrients absorption. Keywords: phytase, Ganoderma sp., optimization, concentration, formulation, additives and spray-drying

Identification of a Proliferation Gene Cluster Associated with HPV E6/E7 Expression Level and Viral DNA Load in Invasive Cervical Carcinoma

Specific HPV DNA sequences are associated with more than 90% of invasive carcinomas of the uterine cervix. Viral E6 and E7 oncogenes are key mediators in cell transformation by disrupting TP53 and RB pathways. To investigate molecular mechanisms involved in the progression of invasive cervical carcinoma, we performed a gene expression study on cases selected according to viral and clinical parameters. Using Coupled Two-Way Clustering and Sorting Points Into Neighbourhoods methods, we identified a Cervical Cancer Proliferation Cluster composed of 163 highly correlated transcripts, many of which corresponded to E2F pathway genes controlling cell proliferation, whereas no primary TP53 targets were present in this cluster. The average expression level of the genes of this cluster was higher in tumours with an early relapse than in tumours with a favourable course (P=0.026). Moreover, we found that E6/E7 mRNA expression level was positively correlated with the expression level of the cluster genes and with viral DNA load. These findings suggest that HPV E6/E7 expression level plays a key role in the progression of invasive carcinoma of the uterine cervix via the deregulation of cellular genes controlling tumour cell proliferation. HPV expression level may thus correspond to a biological marker useful for prognosis assessment and specific therapy of the disease

Background

PROCEEDINGS A rich internet application for remote visualization and collaborative annotation of digital slides in histology and cytolog

Analysis of inducers of xylanase and cellulase activities production by Ganoderma applanatum LPB MR-56

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Endocarditis after Use of Tongue Scraper

We explored the neural substrate of anosognosia for cognitive impairment in Alzheimer's disease (AD). Two hundred nine patients with mild to moderate dementia and their caregivers assessed patients' cognitive impairment by answering a structured questionnaire. Subjects rated 13 cognitive domains as not impaired or associated with mild, moderate, severe, or very severe difficulties, and a sum score was calculated. Two measures of anosognosia were derived. A patient's self assessment, unconfounded by objective measurements of cognitive deficits such as dementia severity and episodic memory impairment, provided an estimate of impaired self-evaluative judgment about cognition in AD. Impaired self-evaluation was related to a decrease in brain metabolism measured with 18F-2-fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET) in orbital prefrontal cortex and in medial temporal structures. In a cognitive model of anosognosia, medial temporal dysfunction might impair a comparison mechanism between current information on cognition and personal knowledge. Hypoactivity in orbitofrontal cortex may not allow AD patients to update the qualitative judgment associated with their impaired cognitive abilities. Caregivers perceived greater cognitive impairments than patients did. The discrepancy score between caregiver's and patient's evaluations, an other measure of anosognosia, was negatively related to metabolic activity located in the temporoparietal junction, consistent with an impairment of self-referential processes and perspective taking in AD

Inhibition of anti-tuberculosis T-lymphocyte function with tumour necrosis factor antagonists

Reactivation of latent Mycobacterium tuberculosis (Mtb) infection is a major complication of anti-tumour necrosis factor (TNF)-α treatment, but its mechanism is not fully understood. We evaluated the effect of the TNF antagonists infliximab (Ifx), adalimumab (Ada) and etanercept (Eta) on anti-mycobacterial immune responses in two conditions: with ex vivo studies from patients treated with TNF antagonists and with the in vitro addition of TNF antagonists to cells stimulated with mycobacterial antigens. In both cases, we analysed the response of CD4(+ )T lymphocytes to purified protein derivative (PPD) and to culture filtrate protein (CFP)-10, an antigen restricted to Mtb. The tests performed were lymphoproliferation and immediate production of interferon (IFN)-γ. In the 68 patients with inflammatory diseases (rheumatoid arthritis, spondylarthropathy or Crohn's disease), including 31 patients with a previous or latent tuberculosis (TB), 14 weeks of anti-TNF-α treatment had no effect on the proliferation of CD4(+ )T lymphocytes. In contrast, the number of IFN-γ-releasing CD4(+ )T lymphocytes decreased for PPD (p < 0.005) and CFP-10 (p < 0.01) in patients with previous TB and for PPD (p < 0.05) in other patients (all vaccinated with Bacille Calmette-Guérin). Treatments with Ifx and with Eta affected IFN-γ release to a similar extent. In vitro addition of TNF antagonists to CD4(+ )T lymphocytes stimulated with mycobacterial antigens inhibited their proliferation and their expression of membrane-bound TNF (mTNF). These effects occurred late in cultures, suggesting a direct effect of TNF antagonists on activated mTNF(+ )CD4(+ )T lymphocytes, and Ifx and Ada were more efficient than Eta. Therefore, TNF antagonists have a dual action on anti-mycobacterial CD4(+ )T lymphocytes. Administered in vivo, they decrease the frequency of the subpopulation of memory CD4(+ )T lymphocytes rapidly releasing IFN-γ upon challenge with mycobacterial antigens. Added in vitro, they inhibit the activation of CD4(+ )T lymphocytes by mycobacterial antigens. Such a dual effect may explain the increased incidence of TB in patients treated with TNF antagonists as well as possible differences between TNF antagonists for the incidence and the clinical presentation of TB reactivation

Outcome in Advanced Ovarian Cancer following an Appropriate and Comprehensive Effort at Upfront Cytoreduction: A Twenty-Year Experience in a Single Cancer Institute

Objectives. The purpose of this retrospective evaluation of advanced-stage ovarian cancer patients was to compare outcome with published findings from other centers and to discuss future options for the management of advanced ovarian carcinoma patients. Methods. A retrospective series of 340 patients with a mean age of 58 years (range: 17–88) treated for FIGO stage III and IV ovarian cancer between January 1985 and January 2005 was reviewed. All patients had primary cytoreductive surgery, without extensive bowel, peritoneal, or systematic lymph node resection, thereby allowing initiation of chemotherapy without delay. Chemotherapy consisted of cisplatin-based chemotherapy in combination with alkylating agents before 2000, whereas carboplatin and paclitaxel regimes were generally used after 1999-2000. Overall survival and disease-free survival were analyzed by the Kaplan-Meier method and the log-rank test. Results. With a mean followup of 101 months (range: 5 to 203), 280 events (recurrence or death) were observed and 245 patients (72%) had died. The mortality and morbidity related to surgery were low. The main prognostic factor for overall survival was postoperative residual disease (P < .0002), while the main prognostic factor for disease-free survival was histological tumor type (P < .0007). Multivariate analysis identified three significant risk factors: optimal surgery (RR = 2.2 for suboptimal surgery), menopausal status (RR = 1.47 for postmenopausal women), and presence of a taxane in the chemotherapy combination (RR = 0.72). Conclusion. These results confirm that optimal surgery defined by an appropriate and comprehensive effort at upfront cytoreduction limits morbidity related to the surgical procedure and allows initiation of chemotherapy without any negative impact on survival. The impact of neoadjuvant chemotherapy to improve resectability while lowering the morbidity of the surgical procedure is discussed