Biotechnological potential of alternative carbon sources for production of pectinases by Rhizopus microsporus var. rhizopodiformis

Fungi collected from Brazilian soil and decomposing plants were screened for pectinase production. R. microsporus var. rhizopodiformis was the best producer and was selected to evaluate the pectic enzyme production under several nutritional and environmental conditions. The pectinase production was studied at 40ºC, under 28 carbon sources-supplemented medium. The inducer effect of several agro-industrial residues such as sugar cane bagasse, wheat flour and corncob on polygalacturonase (PG) activity was 4-, 3- and 2-fold higher than the control (pectin). In glucose-medium, a constitutive pectin lyase (PL) activity was detected. The results demonstrated that R. microsporus produced high levels of PG (57.7 U/mg) and PL (88.6 U/mg) in lemon peel-medium. PG had optimum temperature at 65 ºC and was totally stable at 55 ºC for 90 min. Half-life at 70 ºC was 68 min. These results suggested that the versatility of waste carbon sources utilization by R. microsporus, produce pectic enzymes, which could be useful to reduce production costs and environmental impacts related to the waste disposal.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)(CNPq) Conselho de Desenvolvimento Científico e Tecnológic

Acompanhamento da construçao de obras complementares, integradas no aproveitamento hidroelétrico do baixo sabor

Trabalho final de mestrado para obtenção do grau de mestre em Engenharia Civil na área de Especialização em EdificaçõesO presente relatório de estágio enquadra-se no âmbito do Trabalho Final de Mestrado, do curso de Engenharia Civil, Área de Especialização de Edificações, ministrado no Instituto Superior de Engenharia de Lisboa (ISEL). O Estágio foi realizado entre meados de junho e dezembro 2014, na empreitada de construção do Empreendimento do Aproveitamento Hidroelétrico do Baixo Sabor (AHBS), empreitada adjudicada às empresas Bento Pedroso Construções, SA e Lena – Engenharia e Construções, SA, pela empresa EDP – Gestão da Produção de Energia, SA. Concretamente, o estágio traduziu-se no acompanhamento de processos construtivos no Empreendimento, em particular nas Obras Complementares, onde se destaca a Construção do novo Santuário de Santo Antão, com diversas edificações de apoio, 3 Restabelecimentos a Estradas Nacionais, 1 Restabelecimento a um caminho agrícola e 1 Restabelecimento a um caminho florestal. O estágio na empreitada, baseou-se nas seguintes atividades: • Planear, organizar e dirigir os trabalhos, garantindo a sua execução dentro dos prazos e orçamento; • Gerir as relações com os subempreiteiros, supervisionando os trabalhos; • Gerir equipas de trabalho do ACE, materiais e equipamentos envolvidos nas diversas obras. Quando se iniciou o estágio, a Empreitada já se encontrava na sua fase final.The present report refers to the Master’s Disserttion in the Civil Engineering– Buildings Specialization Area, held at the Lisbon’s Politechnic Engineering Institute (ISEL). The post-graduate training was performed between July and December 2014 within the construction contract of the Baixo Sabor Hydroelectric Project (AHBS), a contract awarded to the companies Bento Pedroso Construções, SA and Lena - Engenharia e Construções, SA, by EDP Gestão da Produção de Energia, SA. Objectively, the post-graduate training tasks were the follow up of construction processes in this Contract, in particular in the Complementary tasks, amongst them, being the new Santo Antão Sanctuary an highlight. Additionally, several supporting buildings, as well as 3 restorations of National Roads, 1 restoration of an agricultural path and 1 restoration of a forest path. The post-graduate training was based on the following activities: • Plan, organize and direct the tasks, ensuring its execution within the deadlines and budget; • Manage relations with subcontractors, supervise the subtasks; • Manage the work teams, materials and equipment involved in the various assignments. When the post-graduate training begins, the Contract is already in its final phase.N/

Extracellular tannase from Emericella nidulans showing hypertolerance to temperature and organic solvents

The filamentous fungus Emericella nidulans (=Aspergillus nidulans) produced high levels of extracellular tannase when grown at 30 degrees C, under agitation (100 rpm), for 24 h in Khanna medium supplemented with tannic acid as carbon source. The enzyme was purified 61-fold, with 30% yield. The molecular mass of the native protein was estimated to be 302 kDa by gel filtration, with a carbohydrate content of 50%. Two protein bands (45.8 and 52 kDa) were observed after 12% SDS-PAGE, suggesting a glycoprotein constituted by three copies of each subunit. The extracellular tannase showed temperature and pH optima of 45 degrees C and 5.0, respectively, and was fully stable in the temperature range of 22-50 degrees C. with a half-life (t(50)) of about 72h at 90 degrees C. The enzyme retained around 80% of control activity when maintained for 60h at pH 4.0 or 5.0. Tannase activity was stimulated by Zn(2+), Hg(2+), Co(2+), and the detergents SDS and Triton X-100. Organic solvents (about 50%, v/v) also increased enzyme activity, particularly isopropanol, acetonitrile, and ethanol. The K(m) and V(max) values were 14.01 mM and 2.63 U mg(-1) protein in the presence of tannic acid; and 4.78 mM and 0.29 U mg(-1) protein in the presence of methyl gallate. For propyl gallate, the V(max) was 0.05 U mg(-1) protein, with K(m) of 7.69 mM; for pyrogallol, the V(max) was 7.40 U mg(-1) protein and the K(m) was 16.94 mM. Crown Copyright (C) 2011 Published by Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

cAMP signaling pathway controls glycogen metabolism in Neurospora crassa by regulating the glycogen synthase gene expression and phosphorylation

The cAMP-PKA signaling pathway plays an important role in many biological processes including glycogen metabolism. In this work we investigated its role in the Neurospora crassa glycogen metabolism control using mutant strains affected in components of the pathway, the cr-1 strain deficient in adenylyl cyclase activity therefore has the PKA pathway not active, and the mcb strain a temperature-sensitive mutant defective in the regulatory subunit of PKA therefore is a strain with constitutively active PKA. We analyzed the expression of the gene encoding glycogen synthase (gsn), the regulatory enzyme in glycogen synthesis as a potential target of the regulation. The cr-1 strain accumulated, during vegetative growth, glycogen levels much higher than the wild type strain indicating a role of the PKA pathway in the glycogen accumulation. The gsn transcript was not increased in this strain but the GSN protein was less phosphorylated "in vitro", and therefore more active, suggesting that the post-translational modification of GSN is likely the main mechanism controlling glycogen accumulation during vegetative growth. Heat shock down-regulates gsn gene transcription in the two mutant strains, as well as in the wild type strain, suggesting that the PKA pathway may not be the only pathway having a direct role in gsn transcription under heat shock. DNA-protein complexes were formed between the STRE motif in the gsn promoter and nuclear proteins from heat-shocked mycelium. However STRE was not able to induce transcription of a reporter gene in Saccharomyces cerevisiae, suggesting that the motif might be involved in a different way of regulation in the N. crassa gene expression under heat shock. The CRE-like DNA elements present in the gsn promoter were shown to be bound by different proteins from the PKA mutant strains. The DNA-protein complexes were observed with proteins from the strains grown under normal condition and under heat shock indicating the functionality of this DNA element. In this work we presented some evidences that the PKA signaling pathway regulates glycogen metabolism in N. crassa in a different way when compared to the well-characterized model of regulation existent in S. cerevisiae. (C) 2009 Elsevier B.V. All rights reserved.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Biochemical properties of an extracellular beta-D-fructofuranosidase II produced by Aspergillus phoenicis under Solid-Sate Fermentation using soy bran as substrate

The filamentous fungus A. phoenicis produced high levels of beta-D-fructofuranosidase (FFase) when grown for 72 hrs under Solid-State Fermentation (SSF), using soy bran moistened with tap water (1:0.5 w/v) as substrate/carbon source. Two isoforms (I and II) were obtained, and FFase II was purified 18-fold to apparent homogeneity with 14% recovery. The native molecular mass of the glycoprotein (12% of carbohydrate content) was 158.5 kDa with two subunits of 85 kDa estimated by SDS-PAGE. Optima of temperature and pH were 55 degrees C and 4.5. The enzyme was stable for more than 1 hr at 50 degrees C and was also stable in a pH range from 7.0 to 8.0. FFase II retained 80% of activity after storage at 4 degrees C by 200 hrs. Dichroism analysis showed the presence of random and beta-sheet structure. A. phoenicis FFase II was activated by Mn(2+), Mg(2+) and Co(2+), and inhibited by Cu(2+), Hg(2+) and EDTA. The enzyme hydrolyzed sucrose, inulin and raffinose. K(d) and V(max) values were 18 mM and 189 U/mg protein using sucrose as substrate.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq

Produção e ação de um pool enzimático de Aspergillus phoenicis com fontes de carbono diferentes

Aspergillus phoenicis is an interesting heat tolerant fungus that can synthesize enzymes with several applications in the food industry due to its great hydrolytic potential. In this work, the fungus produced high enzymatic levels when cultivated on inexpensive culture media consisting of flakes from different origins such as cassava flour, wheat fibre, crushed soybean, agro-industrial wastes, starch, glucose or maltose. Several enzymatic systems were produced from these carbon sources, but amylase was the most evident, followed by pectinase and xylanase. Traces of CMCases, avicelase, lipase, β-xylosidase, β-glucosidase and α-glucosidase activities were also detected. Amylases were produced on rye flakes, starch, oat flakes, corn flakes, cassava flour and wheat fibre. Significant amylolytic levels were produced in the culture medium with glucose or when this sugar was exhausted, suggesting an enzyme in the constitutive form. Cassava flour, rye, oats, barley and corn flakes were also used as substrates in the hydrolytic reactions, aiming to verify the liberation potential of reducing sugars. Corn flakes induced greater liberation of reducing sugars as compared to the others. Thin layer chromatography of the reaction end products showed that the hydrolysis of cassava flour liberated maltooligosaccharides, but cassava flour and corn, rye, oats and barley flakes were hydrolyzed to glucose. These results suggested the presence of glucoamylase and α-amylase as part of the enzymatic pool of A. phoencis

Purification and Partial Characterization of an Exo-polygalacturonase from Paecilomyces variotii Liquid Cultures

An extracellular polygalacturonase (PG) produced from Paecilomyces variotii was purified to homogeneity through two chromatography steps using DEAE-Fractogel and Sephadex G-100. The molecular weight of P. variotii PG was 77,300 Da by gel filtration and SDS-PAGE. PG had isoelectric point of 4.37 and optimum pH 4.0. PG was very stable from pH 3.0 to 6.0. The extent of hydrolysis of different pectins by the purified enzyme was decreased with an increase in the degree of esterification. PG had no activity toward non-pectic polysaccharides. The apparent K (m) and V (max) values for hydrolyzing sodium polypectate were 1.84 mg/mL and 432 A mu mol/min/mg, respectively. PG was found to have temperature optimum at 65 A degrees C and was totally stable at 45 A degrees C for 90 min. Half-life at 55 A degrees C was 50.6 min. Almost all the examined metal cations showed partial inhibitory effects under enzymatic activity, except for Na(+1), K(+1), and Co(+2) (1 mM) and Cu(+2) (1 and 10 mM).Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho de Desenvolvimento Cientifico e Tecnologico (CNPq

Production of xylanase by Aspergilli using alternative carbon sources: application of the crude extract on cellulose pulp biobleaching

The ability of xylanolytic enzymes produced by Aspergillus fumigatus RP04 and Aspergillus niveus RP05 to promote the biobleaching of cellulose pulp was investigated. Both fungi grew for 4-5 days in liquid medium at 40A degrees C, under static conditions. Xylanase production was tested using different carbon sources, including some types of xylans. A. fumigatus produced high levels of xylanase on agricultural residues (corncob or wheat bran), whereas A. niveus produced more xylanase on birchwood xylan. The optimum temperature of the xylanases from A. fumigatus and A. niveus was around 60-70A degrees C. The enzymes were stable for 30 min at 60A degrees C, maintaining 95-98% of the initial activity. After 1 h at this temperature, the xylanase from A. niveus still retained 85% of initial activity, while the xylanase from A. fumigatus was only 40% active. The pH optimum of the xylanases was acidic (4.5-5.5). The pH stability for the xylanase from A. fumigatus was higher at pH 6.0-8.0, while the enzyme from A. niveus was more stable at pH 4.5-6.5. Crude enzymatic extracts were used to clarify cellulose pulp and the best result was obtained with the A. niveus preparation, showing kappa efficiency around 39.6% as compared to only 11.7% for that of A. fumigatus.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientfico e Tecnologico (CNPq