Biological and structural characterization of a new PLA(2) from the Crotalus durissus collilineatus venom

In the present article we report on the biological characterization and amino acid sequence of a new basic Phospholipases A(2) (PLA(2)) isolated from the Crotalus durissus collilineatus venom (Cdcolli F6), which showed the presence of 122 amino acid residues with a pI value of 8.3, molecular mass of 14 kDa and revealed an amino acid sequence identity of 80% with crotalic PLA(2)s such as Mojave B, Cdt F15, and CROATOX. This homology, however, dropped to 50% if compared to other sources of PLA(2)s such as from the Bothrops snake venom. Also, this PLA(2) induced myonecrosis, although this effect was lower than that of BthTx-I or whole crotoxin and it was able to induce a strong blockage effect on the chick biventer neuromuscular preparation, independently of the presence of the acid subunid (crotapotin). The neurotoxic effect was strongly reduced by pre-incubation with heparin or with anhydrous acetic acid and rho-BPB showed a similar reduction. The rho-BPB did not reduce significantly the myotoxic activity induced by the PLA(2), but the anhydrous acetic acid treatment and the pre-incu-bation of PLA(2) with heparin reduced significantly its effects. This protein showed a strong antimicrobial activity against Xanthomonas axonopodis passiflorae (Gram-negative), which was drastically reduced by incubation of this PLA(2) with rho-BPB, but this effect was marginally reduced after treatment with anhydrous acetic acid. Our findings here allow to speculate that basic amino acid residues on the C-terminal and molecular regions near catalytic site regions such as Calcium binding loop or rho-wing region may be involved in the binding of this PLA(2) to the molecular receptor to induce the neurotoxic effect. The bactericidal effect, however, was completely dependent on the enzymatic activity of this protein.

Purification and characterization of the biological effects of phospholipase A(2) from sea anemone Bunodosoma caissarum

Sea anemones contain a variety of biologically active substances. Bunodosoma caissarum is a sea anemone from the Cnidaria phylum, found only in Brazilian coastal waters. The aim of the present work was to study the biological effects of PLA(2) isolated from the sea anemone B. caissarum on the isolated perfused kidney, the arteriolar mesenteric bed and on insulin secretion. Specimens of B. caissarum were collected from the Sao Vicente Channel on the southern coast of the State of São Paulo, Brazil. Reverse phase HPLC analysis of the crude extract of B. caissarum detected three PLA(2) proteins (named BcPLA(2)1, BCPLA(2)2 and BcPLA(2)3) found to be active in B. caissarum extracts. MALDI-TOF mass spectrometry of BcPLA(2)1 showed one main peak at 14.7 kDa. The N-terminal amino acid sequence of BcPLA(2)1 showed high amino acid sequence identity with PLA(2) group III protein isolated from the Mexican lizard (PA23 HELSU, HELSU, PA22 HELSU) and with the honey bee Apis mellifera (PLA(2) and 1POC_A). In addition, BcPLA(2)1 also showed significant overall homology to bee PLA(2). The enzymatic activity induced by native BCPLA(2)1 (20 mu g/well) was reduced by chemical treatment with p-bromophenacyl bromide (p-BPB) and with morin. BcPLA(2)1 strongly induced insulin secretion in presence of high glucose concentration. In isolated kidney, the PLA(2) from B. caissarum increased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate, and sodium, potassium and chloride levels of excretion. BcPLA(2)1, however, did not increase the perfusion pressure on the mesenteric vascular bed. In conclusion, PLA(2), a group III phospholipase isolated from the sea anemone B. caissarum, exerted effects on renal function and induced insulin secretion in conditions of high glucose concentration. (C) 2009 Elsevier Ltd. All rights reserved.

Characterization of Paracoccidioides brasiliensis PbDfg5p, a cell-wall protein implicated in filamentous growth

The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of the most frequent systemic mycosis in Latin America. In humans, infection starts by inhalation of fungal propagules, which reach the pulmonary epithelium and differentiate into the yeast parasitic phase. Here we describe the characterization of a Dfg5p ((d) under bar efective for (f) under bar ilamentous (g) under bar rowth) homologue of P. brasiliensis, a predictable cell wall protein, first identified in Saccharomyces cerevisiae. The protein, the cDNA and genomic sequences were analysed. The cloned cDNA was expressed in Escherichia coli and the purified rPbDfg5p was used to obtain polyclonal antibodies. Immunoelectron microscopy and biochemical studies demonstrated the presence of PbDfg5p in the fungal cell wall. Enzymatic treatments identified PbDfg5p as a beta-glucan linked protein that undergoes N -glycosylation. The rPbDfg5p bound to extracellular matrix components, indicating that those interactions could be important for initial steps leading to P. brasiliensis attachment and colonization of host tissues. The P. brasiliensis dfg5 nucleotide and deduced protein, PbDfg5p, sequences reported in this paper had been submitted to the GenBank database under Accession Nos AY307855 (cDNA) and DQ534495 (genomic). Copyright (C) 2007 John Wiley & Sons, Ltd.

Preparation and characterization of free films of high amylose/pectin mixtures cross-linked with sodium trimetaphosphate

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Molecular cloning and biochemical characterization of a myotoxin inhibitor from Bothrops alternatus snake plasma

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

Functional characterization and target discovery of glycoside hydrolases from the digestome of the lower termite Coptotermes gestroi

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Production and characterization of cellulase-free xylanase from Trichoderma inhamatum

The production of extracellular cellulase-free xylanase from Trichoderma inhamatum was evaluated in liquid Vogel medium with different carbon sources as natural substrates and agricultural or agro-industrial wastes. Optimal production of 244.02 U/mL was obtained with xylan as carbon source, pH 6.0 at 25 degrees C, 120 rpm, and 60-h time culture. Optimal conditions for enzyme activity were 50 degrees C and pH 5.5. Thermal stability of T. inhamatum xylanolytic complex expressed as T(1/2) was 2.2 h at 40 degrees C and 2 min at 50 degrees C. The pH stability was high from 4.0 to 11.0.These results indicate possible employment of such enzymatic complex in some industrial processes which require activity in acid pH, wide-ranging pH stability, and cellulase activity absence.

Production and characterization of glucoamylase from fungus Aspergillus awamori expressed in yeast Saccharomyces cerevisiae using different carbon sources

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

A Novel beta-Glucosidase from Sporidiobolus pararoseus: Characterization and Application in Winemaking

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

Partial characterization of protease from a thermophilic fungus, Thermoascus aurantiacus, and its hydrolytic activity on bovine casein

Proteases are one of the most important groups of industrial enzymes, with considerable application in the food industry. The aim of this work was to study a novel protease produced by the thermophilic fungus, Thermoascus aurantiacus, through solid-state fermentation (SSF). The enzyme acted optimally at pH 5.5 and 60 degrees C it was stable up to 60 degrees C for 1 h and in the pH range 3.0-9.5. To elucidate the enzyme's proteolytic activity, its hydrolytic profile on bovine casein, an important protein in the food industry, was studied by enzymatic hydrolysis on skim milk, analyzed by gel electrophoresis (UREA-PAGE), which clearly showed that the protease does not have the same specificity as bovine chymosin. (c) 2006 Elsevier Ltd. All rights reserved.

Production and partial characterization of serine and metallo peptidases secreted by Aspergillus fumigatus Fresenius in submerged and solid state fermentation

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

Structure, genomic DNA typing, and kinetic characterization of the D allozyme of placental alkaline phosphatase (PLAP/ALPP)

The D allozyme of placental alkaline phosphatase (PLAP) displays enzymatic properties at variance with those of the common PLAP allozymes. We have deduced the amino acid sequence of the PLAP D allele by PCR cloning of its gene, ALPP Two coding substitutions were found in comparison With the cDNA of the common PLAP F allele, i.e., 692C>G and 1352A>G, which translate into a P209R and E429G substitution. A single nucleotide primer extension (SNuPE) assay was developed using PCR primers that enable the amplification of a 1.9 kb PLAP fragment. Extension primers were then used on this PCR fragment to detect the 692C>G and 1352A>G substitution. The SNuPE assay on these two nucleotide substitutions enabled us to distinguish the PLAP F and D alleles from the PLAP S/I alleles. Functional studies on the D allozyme were made possible by constructing and expressing a PLAP D cDNA, i.e., [Arg209, Gly429] PLAP, into wildtype Chinese hamster ovary cells. We determined the k(cat) and K-m, of the PLAP S, F. and D allozymes using the non,physiological substrate p-nitrophenylphosphate at an optimal pH (9.8) as well as two physiological substrates, i.e., pyridoxal-5'-phosphate and inorganic pyrophosphate at physiological pH (7.5). We found that the biochemical properties of the D allozyme of PLAP are significantly different from those of the common PLAP allozymes. These biochemical findings suggest that a suboptimal enzymatic function by the PLAP D allozyme may be the basis for the apparent negative selective pressure of the PLAP D allele. The development of the SNuPE assay will enable us to test the hypothesis that the PLAP D allele is subjected to intrauterine selection by examining genomic DNA from statistically informative population samples. Hum Mutat 19:258-267, 2002. (C) 2002 Wiley-Liss, Inc.

Partial purification and characterization of pectin methylesterase from orange (Citrus sinensis) cv. Pera-rio

The enzyme pectin methylesterase (PME) from orange was extracted and partially purified by filtration on Sephadex G-100. The extraction buffer for orange PME was borate-acetate containing 0.4 M NaCl. Orange PME showed optimum pH at 8.0 and optimum temperature at 50C. The PME enzyme was completely inactivated after 1 min of incubation at 90C. The specific activity increased in the presence of 0.15 M NaCl or 0.025 M Na2SO4, 0.10 M KCl, 0.025 M K2SO4, 0.05 and 0.1 M NH4Cl. Lithium chloride and Li(2)SO(4)inhibited the enzymatic activity at all concentrations studied. The K-m and V(max)value of PME were 0.36 mg/mL and 5.26 mu mol/mL-mg protein, respectively.

A comparative study of protein and enzymatic activity in venoms of some common wasps (Hymenoptera: Vespidae) from São Paulo State

The Hymenoptera Aculeata venoms, with few exceptions, have been poorly studied and characterized. Nevertheless, they have raised increasing interest due to their medical importance, since accidents with these insects are fairly frequent in Brazil and may cause severe allergic reactions. The objectives of the present work were the quantitative characterization of the main allergenic enzymes present in the venom of the species Polybia paulista, Polybia ignobilis, Polistes simillimus, and Agelaia pallipes pallipes through biochemical assays for the determination of total protein content, as well as the level of the enzymatic activity of phospholipase, hyaluronidase, acid phosphatase and esterase. These results, in addition to providing biochemical knowledge about the venom of the species in question, also supply studies that allow phylogenetic inferences among them.

Electropolymerization mechanism in aqueous solution of the oxo-manganese complex biomimicking of the enzymatic center present on the photosystem II

The [Mn4 IVO5(terpy)4(H 2O)2]6+ complex, show great potential for electrode modification by electropolymerization using cyclic voltammetry. The electropolymerization mechanism was based on the electronic transfer between dx2-y2 orbitals of the center metallic and pπ orbital of the ligand, which show great complexity of the system due to orbitals overlap present in octahedral complex of the metal-μ-oxo. The voltammetric behavior both in and after electropolymerization process were also discussed, where the best condition of electropolymerization was observed for low scan rate and 50 potential cycles. A study in ITO/glass electrode for better characterization of polymer was also performed. ©The Electrochemical Society.