Carbohydrate metabolism of Xylella fastidiosa: Detection of glycolytic and pentose phosphate pathway enzymes and cloning and expression of the enolase gene

The objective of this work was to assess the functionality of the glycolytic pathways in the bacterium Xylella fastidiosa. To this effect, the enzymes phosphoglucose isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase of the glycolytic pathway, and glucose 6-phosphate dehydrogenase of the Entner-Doudoroff pathway were studied, followed by cloning and expression studies of the enolase gene and determination of its activity. These studies showed that X. fastidiosa does not use the glycolytic pathway to metabolize carbohydrates, which explains the increased duplication time of this phytopatogen. Recombinant enolase was expressed as inclusion bodies and solubilized with urea (most efficient extractor), Triton X-100, and TCA. Enolase extracted from X. fastidiosa and from chicken muscle and liver is irreversibly inactivated by urea. The purification of enolase was partial and resulted in a low yield. No enzymatic activity was detected for either recombinant and native enolases, aldolase, and glyceraldehyde-3-phosphate dehydrogenase, suggesting that X. fastidiosa uses the Entner-Doudoroff pathway to produce pyruvate. Evidence is presented supporting the idea that the regulation of genes and the presence of isoforms with regulation patterns might make it difficult to understand the metabolism of carbohydrates in X. fastidiosa.

Corn and soybean meal metabolizable energy with the addition of exogenous enzymes for poultry

Two metabolism assays were carried out to determine corn and soybean meal metabolizable energy when enzymes were added. In the first trial, 35 cockerels per studied feedstuff (corn and soybean meal) were distributed in a completely randomized experimental design with four treatments of seven replicates of one bird each. The evaluated treatments were: ingredient (corn and soybean meal) with no enzyme addition, with the addition of an enzyme complex (xylanase, amylase, protease - XAP), xylanase, or phytase. Precise feeding method was used to determine true metabolizable energy corrected for nitrogen balance (TMEn). The use of enzymes did not result in any differences (p>0.05) in soybean meal TMEn, but phytase improved corn TMEn in 2.3% (p=0.004). In the second trial, 280 seven-day-old broiler chicks were distributed in a completely randomized experimental design with seven treatments of five replicates of eight birds each. Treatments consisted of corn with no enzyme addition or with the addition of amylase, xylanase, phytase, XAP complex, XAP+phytase combination, or xylanase/ pectinase/β-glucanase complex (XPBG). Corn was supplemented with macro and trace minerals. Total excreta collection was used to determine apparent metabolizable energy corrected for nitrogen balance (AMEn). Differences were observed (p=0.08) in AMEn and dry matter metabolizability coefficient (p=0.03). The combination of the XAP complex with phytase promoted a 2.11% increase in corn AMEn values, and the remaining enzymes allowed increased between 0.86% and 1.66%.

Effect of mannanoligosaccharides and/or enzymes on antibody titers against infectious bursal and Newcastle disease viruses

O efeito da inclusão de mananoligossacarídeo (MOS) e/ou enzimas em dietas de frangos sobre os títulos de anticorpos contra os vírus das doenças de Gumboro (VDG) e de Newcastle (VDN). Setecentos e cinqüenta aves foram distribuídas em um delineamento experimental inteiramente ao acaso, em arranjo fatorial 2 x 2 + 1, com dois níveis de MOS (0 e 0,1% até 21 dias e 0,05% de 22 até 42 dias de idade), dois níveis de enzimas (0 e 0,05%) e uma dieta-controle-positivo contendo antibióticos, totalizando cinco tratamentos com cinco repetições. Para análise dos anticorpos, amostras de sangue foram colhidas semanalmente por punção da veia jugular em duas aves de cada repetição. A primeira e a última colheita foram realizadas aos sete e 42 dias de idade, respectivamente. A inclusão de MOS resultou em aumento dos títulos contra VDG na quarta (P<0,03) e quinta (P<0,02) semanas, e contra VDN na terceira (P<0,01), quarta (P<0,03) e quinta (P<0,03) semanas de idade. O MOS foi efetivo em estimular a resposta imune humoral contra VDG e VDN vacinais.

Performance and morphology of intestinal mucosa of broilers fed mannan-oligosaccharides and enzymes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Production of fibrolytic enzymes by Aspergillus japonicus C03 using agro-industrial residues with potential application as additives in animal feed

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of Aspergillus spp. exogenous fibrolytic enzymes on in vitro fermentation of tropical forages

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação, identificação e atividade enzimática de bactérias psicotróficas presentes no leite cru refrigerado

Extended storage of refrigerated milk can lead to reduced quality of raw and processed milk, which is a consequence of the growth and metabolic activities of psychrotrophic bacteria, able to grow under 7oC or lower temperatures. Although most of these microorganisms are destroyed by heat treatment, some have the potential to produce termoresistant proteolytic and lipolytic enzymes that can survive even UHT processing and reduce the processed products quality. Recently, the IN 51 determineds that milk should be refrigerated and stored at the farm what increased the importance of this group of microorganisms. In this work, psychrotrophic bacteria were isolated from 20 communitarian bulk tanks and 23 individual bulk tanks from dairy farms located at Zona da Mata region of Minas Gerais State and from southeastern Rio de Janeiro. Selected milk dilutions were plated on standard agar and after incubation for 10 days at 7oC, five colonies were isolated, firstly using nutrient agar and after using McConkey agar for 24 hours at 21oC. The isolates were identified by morphology, Gram stain method, catalase production, fermentative/oxidative metabolism and by API 20E, API 20NE, API Staph, API Coryne or API 50 CH (BioMerieux). In order to ensure reproductibility, API was repeated for 50% of the isolates. Species identification was considered when APILAB indexes reached 75% or higher. 309 strains were isolated, 250 Gram negative and 59 Gram positive. 250 Gram negative isolates were identified as: Acinetobacter spp. (39), Aeromonas spp. (07), A. Hydrophila (16), A. sobria (1), A. caviae (1), Alcaligenes feacalis (1), Burkholderia cepacia (12), Chryseomonas luteola (3), Enterobacter sp. (1), Ewingella americana(6), Hafnia alvei (7), Klebsiella sp. (1), Klebsiella oxytoca (10), Yersinia spp. (2), Methylobacterium mesophilicum (1), Moraxella spp. (4), Pantoea spp. (16), Pasteurella sp. (1), Pseudomonas spp. (10), P. fluorescens (94), P. putida (3), Serratia spp. (3), Sphigomonas paucomobilis (1). Five isolates kept unidentified. Pseudomonas was the predominant bacteria found (43%) and P. fluorescens the predominant species (37.6%), in accordance with previous reports. Qualitative analysis of proteolytic and lipolytic activity was based on halo formation using caseinate agar and tributirina agar during 72 hours at 21oC and during 10 days at 4°C, 10oC and 7°C. Among 250 Gram negative bacteria found, 104 were identified as Pseudomonas spp. and 60,57% of this group showed proteolytic and lipolytic acitivities over all four studied temperatures. 20% of Acinetobacter, Aeromonas, Alcaligenes, Burkholderia, Chryseomonas, Methylobacterium, Moraxella presented only lipolytic activity. Some isolates presented enzymatic activity in one or more studied temperatures. Among Gram positive bacteria, 30.51% were proteolytic and lipolytic at 10oC, 8.47% were proteolytic at 7oC, 10oC, and 21oC, 8.47% were proteolytic at all studied temperatures (4oC, 7oC, 10oC and 21oC) and 3.38% were proteolytic only at 21oC. At 4oC, only one isolate showed proteolytic activity and six isolates were lipolytic. In relation to Gram negative microorganisms, 4% were proteolytic and lipolytic at 7oC, 10oC and 21oC, 10% were proteolytic at 10oC and 4.4% were lipolytic at 4oC, 7oC, 10oC and 21oC, while 6.4% of all isolates were proteolytic and lipolytic at 10oC and 21oC as well as lipolytic at 4oC and 7oC. These findings are in accordance with previous researches that pointed out Pseudomonas as the predominant psycrotrophic flora in stored refrigerated raw milk

Production of active recombinant eIF5A: reconstitution in E.coli of eukaryotic hypusine modification of eIF5A by its coexpression with modifying enzymes

Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the polyamine-modified lysine, hypusine [N(epsilon)-(4-amino-2-hydroxybutyl)lysine]. Hypusine occurs only in eukaryotes and certain archaea, but not in eubacteria. It is formed post-translationally by two consecutive enzymatic reactions catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). Hypusine modification is essential for the activity of eIF5A and for eukaryotic cell proliferation. eIF5A binds to the ribosome and stimulates translation in a hypusine-dependent manner, but its mode of action in translation is not well understood. Since quantities of highly pure hypusine-modified eIF5A is desired for structural studies as well as for determination of its binding sites on the ribosome, we have used a polycistronic vector, pST39, to express eIF5A alone, or to co-express human eIF5A-1 with DHS or with both DHS and DOHH in Escherichia coli cells, to engineer recombinant proteins, unmodified eIF5A, deoxyhypusine- or hypusine-modified eIF5A. We have accomplished production of three different forms of recombinant eIF5A in high quantity and purity. The recombinant hypusine-modified eIF5A was as active in methionyl-puromycin synthesis as the native, eIF5A (hypusine form) purified from mammalian tissue. The recombinant eIF5A proteins will be useful tools in future structure/function and the mechanism studies in translation.

Effect of oral vanadyl sulfate treatment on serum enzymes and lipids of streptozotocin-diabetic young rats

In this work we investigate the possible toxicity of vanadyl sulfate (VOSO4), a compound capable of reducing hyperglycemia, on the following serum enzymes of diabetic young rats: alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LD) and creatine kinase (CK), as well as its effects on serum lipids. We find that at a concentration of 1 mg/mL VOSO4 has no toxic effect on the liver and muscles of diabetics young rats. These findings suggest that VOSO4 may be an alternative to insulin in the near future, due to its low cost, low toxicity and ready availability.

Caracterização e hidrólise in vitro da globulina principal de grão-de-bico (Cicer arietinum L.), var. IAC-Marrocos

No presente estudo procedeu-se ao isolamento e caracterização da fração globulina majoritária (11 S) de grão-de-bico, var. IAC-Marrocos. A globulina majoritária extraída foi isolada por cromatografia de filtração em gel e de troca-iônica mostrando apenas uma banda de proteína na eletroforese em gel de poliacrilamida. A globulina majoritária, após passagem em coluna de Sephadex, revelou duas bandas protéicas de 55 e 52,5kDa e três bandas menores em gel de poliacrilamida dodecilsulfato de sódio. Na presença de 2-mercaptoetanol 6 polipeptídios na faixa de 18 a 42kDa foram revelados na eletroforese. A globulina isolada foi submetida à ação da tripsina e quimotripsina onde a forma nativa mostrou-se resistente à ação enzimática enquanto o aquecimento (96 e 121°C/15min) não foi suficiente para aumentar a susceptibilidade à hidrólise, significativamente. Adição de NaCl 0,3M levou a um aumento da estabilidade estrutural com menor susceptibilidade à digestão proteolítica, fato em parte perdido com o aquecimento. As hidrólises foram acompanhadas por eletroforese em gel de poliacrilamida dodecilsulfato de sódio.

High Incidence of Enterotoxin D Producing Staphylococcus spp. in Brazilian Cow's Raw Milk and Its Relation with Coagulase and Thermonuclease Enzymes

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Molecular characterization of BjussuSP-I, a new thrombin-like enzyme with procoagulant and kallikrein-like activity isolated from Bothrops jararacussu snake venom

A thrombin-like enzyme, named BjussuSP-I, isolated from Bothrops jararacussu snake venom, is an acidic single-chain glycoprotein with M-r = 61,000, pI similar to 3.8 and 6% sugar. BjussuSP-I shows high proteolytic activity upon synthetic substrates, such as S-2238 and S-2288. It also shows procoagulant and kallikrein-like activity, but is unable to act on platelets and plasmin. These activities are inhibited by specific inhibitors of this class of enzymes. The complete cDNA sequence of BjussuSP-I with 696 bp encodes open reading frames of 232 amino acid residues, which conserve the common domains of thrombin-like serine proteases. BjussuSP-I shows a high structural homology with other thrombin-like enzymes from snake venoms where common amino acid residues are identified as those corresponding to the catalytic site and subsites S1, S2 and S3 already reported. In this study, we also demonstrated the importance of N-linked glycans, to improve thrombin-like activity of BjussuSP-I toxin. (c) 2007 Elsevier Masson SAS. All rights reserved.

Messenger ribonucleic acid abundance of intestinal enzymes and transporters in feed-restricted and refed chickens at different ages

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)