Purification and characterization of two beta-glucosidases from the thermophilic fungus Thermoascus aurantiacus

beta-Glucosidase from the fungus Thermoascus aurantiacus grown oil semi-solid fermentation medium (using ground corncob as substrate) was partially purified in 5 steps - ultrafiltration, ethanol precipitation, gel filtration and 2 anion exchange chromatography runs, and characterized. After the first anion exchange chromatography, beta-glucosidase activity was eluted in 3 peaks (Gl-1, Gl-2, Gl-3). Only the Gl-2 and Gl-3 fractions were adsorbed on the gel matrix. Gl-2 and Gl-3 exhibited optimum pH at 4.5 and 4.0, respectively. The temperature optimum of both glucosidases was at 75-80 degreesC. The pH stability of Gl-2 (4.0-9.0) was higher than Gl-3 (5.5-8.5); both enzyme activities showed similar patterns of thermostability. Under conditions of denaturing gel chromatography the molar mass of Gl-2 and Gl-3 was 175 and 157 kDa, respectively. Using 4-nitrophenyl beta-D-glucopyranoside as substrate, K-m, values of 1.17 +/- 0.35 and 1.38 +/- 0.86 mmol/L were determined for Gl-2 and Gl-3, respectively. Both enzymes were inhibited by Ag+ and stimulated by Ca2+.

Partial purification and characterization of lysine-ketoglutarate reductase in normal and opaque-2 maize endosperms

Lysine-ketoglutaratc reductase catalyzes the first step of lysine catabolism in maize (Zea mays L.) endosperm. The enzyme condenses L-lysine and α-ketoglutarate into saccharopine using NADPH as cofactor. It is endosperm-specific and has a temporal pattern of activity, increasing with the onset of kernel development, reaching a peak 20 to 25 days after pollination, and thereafter decreasing as the kernel approaches maturity. The enzyme was extracted from the developing maize endosperm and partially purified by ammonium-sulfate precipitation, anion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Blue-Sepharose CL-6B. The preparation obtained from affinity chromatography was enriched 275-fold and had a specific activity of 411 nanomoles per minute per milligram protein. The native and denaturated enzyme is a 140 kilodalton protein as determined by polyacrylamide gel electrophoresis. The enzyme showed specificity for its substrates and was not inhibited by either aminoethyl-cysteine or glutamate. Steady-state product-inhibition studies revealed that saccharopine was a noncompetitive inhibitor with respect to α-ketoglutarate and a competitive inhibitor with respect to lysine. This is suggestive of a rapid equilibriumordered binding mechanism with a binding order of lysine, α-ketoglutarate, NADPH. The enzyme activity was investigated in two maize inbred lines with homozygous normal and opaque-2 endosperms. The pattern of lysine-ketoglutarate reductase activity is coordinated with the rate of zein accumulation during endosperm development. A coordinated regulation of enzyme activity and zein accumulation was observed in the opaque-2 endosperm as the activity and zein levels were two to three times lower than in the normal endosperm. Enzyme extracted from L1038 normal and opaque-2 20 days after pollination was partially purified by DEAE-cellulose chromatography. Both genotypes showed a similar elution pattern with a single activity peak eluted at approximately 0.2 molar KCL. The molecular weight and physical properties of the normal and opaque-2 enzymes were essentially the same. We suggest that the Opaque-2 gene, which is a transactivator of the 22 kilodalton zein genes, may be involved in the regulation of the lysine-ketoglutarate reductase gene in maize endosperm. In addition, the decreased reductase activity caused by the opaque-2 mutation may explain, at least in part, the elevated concentration of lysine found in the opaque-2 endosperm.

Níveis críticos de fósforo no solo e nas folhas para a implantação de Eucalyptus grandis Hill ex Maiden, em quatro tipos de solos

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

Mecanismos de absorção de aminoácidos e oligopeptídios. Controle e implicações na dietoterapia humana

The mechanisms involved in the absorption of amino acids and oligopeptides are reviewed regarding their implications in human feedings. Brush border and basolateral membranes are crossed by amino acids and di-tripeptides by passive (facilitated or simple diffusion) or active (Na + or H + co-transporters) pathways. Active Na +-dependent system occurs mainly at brush border and simple diffusion at basolateral, both membranes have the passive facilitated transport. Free-amino acids use either passive or active transport systems whereas di-tripeptides do mainly active (H + co-transporter). Brush border have distinctive transport system for amino acids and di-tripeptides. The former occurs mainly by active Na + dependency whereas the later is active H +-dependent with little affinity for tetra or higher peptides. Free amino acids are transported at different speed by saturable, competitive carriers with specificity for basic, acidic or neutral amino acids. Di and tripeptides have at least two carriers both electrogenic and H +-dependent. The basolateral membrane transport of amino acids is mostly by facilitated diffusion while for di-tripeptides it is an active anion exchange associated process. The main regulation of amino acids and di-tripeptide transport is the presence o substrate at the mucosal membrane with higher the substrate higher the absorption. Di and tripeptides are more efficiently absorbed than free amino acids which in turns are better absorbed than oligopeptides. So di-tripeptides result in better N-retention and is particularly useful in cases of lower intestinal absorption capacity. The non-absorbed peptides are digested and fermented by colonic bacteria resulting short-chain fatty acids, dicarboxylic acids, phenolic compounds and ammonia. Short-chain fatty acid provides energy for colonocytes and bacteria and the ammonia not fixed by bacteria returns to the liver for ureagenesis.

Purification and characterization of two β-glucosidases from the thermophilic fungus Thermoascus aurantiacus

β-Glucosidase from the fungus Thermoascus aurantiacus grown on semi-solid fermentation medium (using ground corncob as substrate) was partially purified in 5 steps-ultrafiltration, ethanol precipitation, gel filtration and 2 anion exchange chromatography runs, and characterized. After the first anion exchange chromatography, β-glucosidase activity was eluted in 3 peaks (Gl-1, Gl-2, Gl-3). Only the Gl-2 and Gl-3 fractions were adsorbed on the gel matrix. Gl-2 and Gl-3 exhibited optimum pH at 4.5 and 4.0, respectively. The temperature optimum of both glucosidases was at 75-80°C. The pH stability of Gl-2 (4.0-9.0) was higher than Gl-3 (5.5-8.5); both enzyme activities showed similar patterns of thermostability. Under conditions of denaturing gel chromatography the molar mass of Gl-2 and Gl-3 was 175 and 157 kDa, respectively. Using 4-nitrophenyl β-D-glucopyranoside as substrate, Km values of 1.17 ± 0.35 and 1.38 ± 0.86 mmol/L were determined for Gl-2 and Gl-3, respectively. Both enzymes were inhibited by Ag+ and stimulated by Ca2+.

One-step purification of 5-enolpyruvylshikimate-3-phosphate synthase enzyme from Mycobacterium tuberculosis

Currently, there are 8 million new cases and 2 million deaths annually from tuberculosis, and it is expected that a total of 225 million new cases and 79 million deaths will occur between 1998 and 2030. The reemergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons, and the proliferation of multi-drug-resistant strains have created a need to develop new antimycobacterial agents. The existence of homologues to the shikimate pathway enzymes has been predicted by the determination of the genome sequence of Mycobacterium tuberculosis. We have previously reported the cloning and overexpression of M. tuberculosis aro A-encoded EPSP synthase in both soluble and active forms, without IPTG induction. Here, we describe the purification of M. tuberculosis EPSP synthase (mtEPSPS) expressed in Escherichia coli BL21(DE3) host cells. Purification of mtEPSPS was achieved by a one-step purification protocol using an anion exchange column. The activity of the homogeneous enzyme was measured by a coupled assay using purified shikimate kinase and purine nucleoside phosphorylase proteins. A total of 53 mg of homogeneous enzyme could be obtained from 1 L of LB cell culture, with a specific activity value of approximately 18 U mg-1. The results presented here provide protein in quantities necessary for structural and kinetic studies, which are currently underway in our laboratory. © 2002 Elsevier Science (USA). All rights reserved.

Efeitos do fósforo do solo no desenvolvimento inicial de Genipa americana L.

A greenhouse experiment was carried out to evaluate the effects of soil phosphorus in the development of plants of Genipa americana L. Five doses of P (0, 50, 100, 200, and 400 mg dm -3) were applied in pots with 10 dm 3 of three Neossolos Quartzarênicos (NQ 1, NQ 2 and NQ 3) and one Latossolo Vermelho (LV), that were used in an experiment with Eucalyptus plants during three months. After this period, the soils were analyzed and a 30 cm height seedling of G. americana was planted in each pot. The experiment design was completely randomized, in 5 × 4 factorial scheme, with five replications. The largest biomass of the stem was obtained with 45 mg dm -3 of P in the soil NQ 1 (9% of clay) and 59 mg dm -3 in the soil NQ 3 (14% of clay). In the LV (40% of clay), the biomass of the stem increased linearly in the band of 3 to 148 mg dm -3 of P. The phosphorus critical level in the soil for 80% of the steam and leaves dry matter production of G. americana at planting phase is 15 mg dm -3, using an anion-exchange resin as extractor.

cDNA cloning and 1.75 Å crystal structure determination of PPL2, an endochitinase and N-acetylglucosamine-binding hemagglutinin from Parkia platycephala seeds

Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) seeds by affinity chromatography and RP-HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407 ± 15 Da, which contains six cysteine residues engaged in the formation of three intramolecular disulfide bonds. Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was specifically inhibited by N-acetylglucosamine. In addition, Parkia platycephala lectin 2 hydrolyzed β(1-4) glycosidic bonds linking 2-acetoamido-2-deoxy-β-d-glucopyranose units in chitin. The full-length amino acid sequence of Parkia platycephala lectin 2, determined by N-terminal sequencing and cDNA cloning, and its three-dimensional structure, established by X-ray crystallography at 1.75 Å resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which share the (βα) 8 barrel topology harboring the catalytic residues Asp125, Glu127, and Tyr182. © 2006 The Authors.

Avaliação de extratores químicos na determinação de silício disponível em solos cultivados com cana-de-açúcar

Pós-graduação em Agronomia (Energia na Agricultura) - FCA

Análise de especiação redox de arsênio in situ usando a técnica de difusão de filmes finos por gradiente de concentração

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Caracterização físico-química de pectinases extracelulares purificadas de Aspergillus giganteus

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

Mineralização de nitrogênio de solos e de resíduo orgânico em laboratório e em campo

Pós-graduação em Agronomia (Ciência do Solo) - FCAV

Purificação e caracterização bioquímica da α-glicosidase termoestável produzida por Thermoascus aurantiacus CBMAI 756

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

Uranium-lead dating method at the Pará-Iso isotope geology laboratory, UFPA, Belém – Brazil

ABSTRACT: Analytical procedures for U-Pb isotope dilution analyses at the Pará-Iso isotope geology laboratory of the Federal University of Pará (UFPA) are described in detail. The procedures are applied to zircon, titanite, rutile, apatite, columbite-tantalite and whole rock. Reagent preparation and chemical processing are done in clean-room conditions. Samples are dissolved using Teflon™ microcapsules in steel jacket Teflon™ Parr Instrument™ bomb or Teflon™ screw cap containers. U and Pb are separated using anion exchange AG 1x8 resin columns. Typical blanks for mineral sample amounts of 0.01 to 1.0 mg are less than 1 pg U and 20-30 pg Pb. Isotope analysis of the U and Pb from the same filament are carried out using a Finnigan MAT 262 mass-spectrometer in static and dynamic modes. The current analytical level is demonstrated on analyses of international standard zircon 91500 with three different ²³⁵U-²⁰⁵Pb and ²³⁵U-²⁰⁸Pb isotope tracers and whole rock standards. Results of analyses of two zircon samples are also presented.

Síntese e caracterização de hidróxidos duplos a partir da lama vermelha

O resíduo do refino da bauxita, comumente conhecido como lama vermelha, tem mostrado ser capaz de ligar metais traços tais como o cobre, cádmio, zinco, cromo, níquel e chumbo sob condições apropriadas. A neutralização da lama vermelha é necessária porque essa lama é tipicamente cáustica, com pH em torno de 13. Muitas pesquisas têm sido desenvolvidas envolvendo a utilização da lama vermelha, porque contem uma série de elementos valiosos. Assim a utilização da lama vermelha como material de construção e como adsorvente é atrativa porque ela é utilizada em grande quantidade. Nada pode ser feito sem descartar componentes valiosos como titânio, zircônio e principalmente ferro. A síntese de hidrotalcita conectada com a reciclagem de soluções alcalinas representa uma maneira efetiva de uso dessa solução considerando a forte demanda por proteção ambiental no mundo. Os compostos tipo hidrotalcita, precipitados durante a neutralização da lama, também removem oxi-ânions de metais de transição através da combinação de intercalações e adsorção de espécies aniônicas na sua superfície externa. Os hidróxidos duplos lamelares (HDL) vêm sendo investigados à muitos anos como materiais hospedeiros para uma variedade de reações de intercalação de anions. A estrutura lamelar dos HDLs pode ser usada para controlar a adição ou remoção de uma variedade de espécies químicas, tanto orgânicas quanto inorgânicas. Isto é alcançado através de sua habilidade de adaptar a separação das lamelas de hidróxidos, e da reatividade da região interlamelar. O material resultante adsorve anions quando colocado em soluções e reverte a estrutura da hidrotalcita. Avanços significativos têm sido alcançados recentemente na caracterização desses materiais, incluindo estudos estruturais sobre o mecanismo de intercalação. O objetivo desse trabalho foi a síntese e caracterização de HDLs a partir da lama vermelha, e testar esses materiais como adsorventes de metais pesados.