Aspectos comparativos das luciferases pH-insensitivas de Pyrearinus termitilluminans (Coleoptera: Elateridae) e Phrixotrix spp (Coleoptera: Phengodidae): expressão, purificação e propriedades do sítio ativo

Pós-graduação em Ciências Biológicas (Biologia Celular e Molecular) - IBRC

Sinergismo entre sulfito, ácido lático, PH e etanol na fermentação alcoólica de Saccharomyces cerevisiae PE-2 e M-26

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Culture of osteogenic cells from human alveolar bone: A useful source of alkaline phosphatase

The aim of this study was to obtain membrane-bound alkaline phosphatase from osteoblastic-like cells of human alveolar bone. Cells were obtained by enzymatic digestion and maintained in primary culture in osteogenic medium until subconfluence. First passage cells were cultured in the same medium and at 7, 14, and 21 days, total protein content, collagen content, and alkaline phosphatase activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Cells in primary culture at day 14 were washed with Tris-HCl buffer, and used to extract the membrane-bound alkaline phosphatase. Cells expressed osteoblastic phenotype. The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10.0. This enzyme also hydrolyzes ATP, ADP, fructose-1-phosphate, fructose-6-phosphate, pyrophosphate and beta-glycerophosphate. PNPPase activity was reduced by typical inhibitors of alkaline phosphatase. SDS-PAGE of membrane fraction showed a single band with activity of similar to 120 kDa that could be solubilized by phospholipase C or Polidocanol. (c) 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture

Cells from rat bone marrow exhibit the proliferation-differentiation sequence of osteoblasts, form mineralized extracellular matrix in vitro and release alkaline phosphatase into the medium. Membrane-bound alkaline phosphatase was obtained by method that is easy to reproduce, simpler and fast when compared with the method used to obtain the enzyme from rat osseous plate. The membrane-bound alkaline phosphatase from cultures of rat bone marrow cells has a MWr of about 120 kDa and specific PNPP activity of 1200 U/tng. The ecto-enzyme is anchored to the plasma membrane by the GPI anchor and can be released by PIPLC (selective treatment) or polidocanol (0.2 mg/mL protein and 1% (w/v) detergent). The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10. This fraction hydrolyzes ATP (240 U/mg), ADP (350 U/ mg), glucose 1-phosphate (1100 U/mg), glucose 6-phosphate (340 Wing), fructose 6-phosphate (460 U/mg), pyrophosphate (330 U/mg) and (3glycerophosphate (600 U/mg). Cooperative effects were observed for the hydrolysis of PPi and beta-glycerophosphate. PNPPase activity was inhibited by 0.1 mM vanadate (46%), 0.1 mM ZnCl2 (68%), 1 mM levamisole (66%), 1 mM arsenate (44%), 10 mM phosphate (21%) and 1 mM theophylline (72%). We report the biochemical characterization of membrane-bound alkaline phosphatase obtained from rat bone marrow cells cultures, using a method that is simple, rapid and easy to reproduce. Its properties are compared with those of rat osseous plate enzyme and revealed that the alkaline phosphatase obtained has some kinetics and structural behaviors with higher levels of enzymatic activity, facilitating the comprehension of the mineralization process and its function. (c) 2006 Elsevier B.V. All rights reserved.

Kinetic characterization of a membrane-specific ATPase from rat osseous plate and its possible significance on endochondral ossification

Treatment with phosphatidylinositol-specific phospholipase C of rat osseous plate membranes released up to 90-95% of alkaline phosphatase, but a specific ATPase activity (optimum pH = 7.5) remained bound to the membrane. The hydrolysis of ATP by this ATPase was negligible in the absence of magnesium or calcium ions. However, at millimolar concentrations of magnesium and calcium ions, the membrane-specific ATPase activity increased to about 560-600 U/mg, exhibiting two classes of ATP-hydrolysing sites, and site-site interactions. GTP, UTP, ITP, and CTP were also hydrolyzed by the membrane-specific ATPase. Oligomycin, ouabain, bafilomycin A(1), thapsigargin, omeprazole, ethacrynic acid and EDTA slightly affected membrane-specific ATPase activity while vanadate produced a 18% inhibition. The membrane-specific ATPase activity was insensitive to theophylline, but was inhibited 40% by levamisole. These data suggested that the membrane-specific ATPase activity present in osseous plate membranes, and alkaline phosphatase, were different proteins. (C) 1998 Elsevier B.V. B.V.

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)

Properties of a new fungal b-galactosidase with potential application in the dairy industry

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

Production and action pattern of inulinase from Aspergillus Niger-245: hydrolysis of inulin from several sources

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

Purification and characterization of β-Fructosidase with inulinase activity from Aspergillus niger - 245

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

Polyphenol oxidase from yacon roots (Smallanthus sonchifolius)

Polyphenol oxidase (E.C. 1.14.18.1) (PPO) extracted from yacon roots (Smallanthus sonchifolius) was partially purified by ammonium sulfate fractionation and separation on Sephadex G-100. The enzyme had a molecular weight of 45 490 +/- 3500 da and K-m values of 0.23, 1.14, 1.34, and 5.0 mM for the substrates caffeic acid, chlorogenic acid, 4-methylcatechol, and catechol, respectively. When assayed with resorcinol, DL-DOPA, pyrogallol, protocatechuic, p-coumaric, ferulic, and cinnamic acids, catechin, and quercetin, the PPO showed no activity. The optimum pH varied from 5.0 to 6.6, depending on substrate. PPO activity was inhibited by various phenolic and nonphenolic compounds. p-Coumaric and cinnamic acids showed competitive inhibition, with K-i values of 0.017 and 0.011 mM, respectively, using chlorogenic acid as substrate. Heat inactivation from 60 to 90 degrees C showed the enzyme to be relatively stable at 60-70 degrees C, with progressive inactivation when incubated at 80 and 90 degrees C. The E-a (apparent activation energy) for inactivation was 93.69 kJ mol(-1). Sucrose, maltose, glucose, fructose, and trehalose at high concentrations appeared to protect yacon PPO against thermal inactivation at 75 and 80 degrees C.

POLYPHENOLOXIDASE FROM ATEMOYA FRUIT (ANNONA CHERIMOLA MILL. ANNONA SQUAMOSA L.)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Some Biochemical properties of polyphenoloxidase from spearmint (Mentha arvensis)

Polifenoloxidase (PPO, EC 1.14.18.1) extraída de folhas de Mentha arvensis foi isolada por precipitação com (NH4)2SO4 e diálise extensiva. Seu pH e temperatura ótimos variaram com o substrato. A PPO apresentou atividade com vários difenóis. Valores de Km foram 0,825; 0,928 e 7,41 mM para ácido caféico, 4-metilcatecol e catecol, respectivamente. Na inativação térmica, 50% da enzima foi inativada após 60 e 15 segundos a 70 e 75ºC, respectivamente. A medida de atividade residual mostrou um efeito estabilizante de sacarose a várias temperaturas e uma energia de ativação (Ea) para inativação aumentando com a concentração de sacarose de 0 a 40% (p/p). Valores de energias de ativação de 78,13; 80,37; 82,79 and 81,00 kJ/Mol foram encontradas para 0, 15, 30 e 40% de sacarose, respectivamente. A PPO foi inibida pelos ácidos ascórbico, benzóico, cinamico, ferulico, p-cumárico, protocatéquico, além de metabisulfito de sódio, resorcinol e pirogalol. Os valores de Ki mostram que o ácido ascórbico foi o mais efetivo inibidor. O tipo de inibição foi determinado para cada inibidor.

Screening and Production Study of Microbial Xylanase Producers from Brazilian Cerrado

Hemicelluloses are polysaccharides of low molecular weight containing 100 to 200 glycosidic residues. In plants, the xylans or the hemicelluloses are situated between the lignin and the collection of cellulose fibers underneath. The xylan is the most common hemicellulosic polysaccharide in cell walls of land plants, comprising a backbone of xylose residues linked by beta-1,4-glycosidic bonds. So, xylanolytic enzymes from microorganism have attracted a great deal of attention in the last decade, particularly because of their biotechnological characteristics in various industrial processes, related to food, feed, ethanol, pulp, and paper industries. A microbial screening of xylanase producer was carried out in Brazilian Cerrado area in Selviria city, Mato Grosso do Sul State, Brazil. About 50 bacterial strains and 15 fungal strains were isolated from soil sample at 35 A degrees C. Between these isolated microorganisms, a bacterium Lysinibacillus sp. and a fungus Neosartorya spinosa as good xylanase producers were identified. Based on identification processes, Lysinibacillus sp. is a new species and the xylanase production by this bacterial genus was not reported yet. Similarly, it has not reported about xylanase production from N. spinosa. The bacterial strain P5B1 identified as Lysinibacillus sp. was cultivated on submerged fermentation using as substrate xylan, wheat bran, corn straw, corncob, and sugar cane bagasse. Corn straw and wheat bran show a good xylanase activity after 72 h of fermentation. A fungus identified as N. spinosa (strain P2D16) was cultivated on solid-state fermentation using as substrate source wheat bran, wheat bran plus sawdust, corn straw, corncob, cassava bran, and sugar cane bagasse. Wheat bran and corncobs show the better xylanase production after 72 h of fermentation. Both crude xylanases were characterized and a bacterial xylanase shows optimum pH for enzyme activity at 6.0, whereas a fungal xylanase has optimum pH at 5.0-5.5. They were stable in the pH range 5.0-10.0 and 5.5-8.5 for bacterial and fungal xylanase, respectively. The optimum temperatures were 55C and 60 A degrees C for bacterial and fungal xylanase, respectively, and they were thermally stable up to 50 A degrees C.

Evaluation of solid and submerged fermentations for the production of cyclodextrin glycosyltransferase by Paenibacillus campinasensis H69-3 and characterization of crude enzyme

Cyclodextrin glycosyltransferase (CGTase) is an enzyme that produces cyclodextrins from starch by an intramolecular transglycosylation reaction. Cyclodextrins have been shown to have a number of applications in the food, cosmetic, pharmaceutical, and chemical industries. In the current study, the production of CGTase by Paenibacillus campinasensis strain H69-3 was examined in submerged and solid-state fermentations. P. campinasensis strain H69-3 was isolated from the soil, which grows at 45 C, and is a Gram-variable bacterium. Different substrate sources such as wheat bran, soybean bran, soybean extract, cassava solid residue, cassava starch, corn starch, and other combinations were used in the enzyme production. CGTase activity was highest in submerged fermentations with the greatest production observed at 48-72 h. The physical and chemical properties of CGTase were determined from the crude enzyme produced from submerged fermentations. The optimum temperature was found to be 70-75 degrees C, and the activity was stable at 55 degrees C for 1 h. The enzyme displayed two optimum pH values, 5.5 and 9.0 and was found to be stable between a pH of 4.5 and 11.0.

Production of extracellular acid proteases by Aspergillus clavatus

The production of extracellular acid proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on haemoglobin pH 5.0 at 37 degreesC. The highest acid proteolytic activity (80 U/ml) was observed in culture medium containing glucose and gelatin at 1% (w/v) at 30 degreesC at the third day of incubation. Cultures developed in Vogel medium with glucose at 2% (w/v) showed at about 45% of proteolytic activity when compared to the cultures with 1% of the same sugar. The optimum pH of enzymatic activity was 2.0 and the enzyme was stable at pH values ranging from 2.0 to 4.0. The optimum temperature was 40 degreesC and the half-lives at 40, 45 and 50 degreesC were 30, 10 and 5 min, respectively.