Production and characterization of a milk-clotting protease in the crude enzymatic extract from the newly isolated Thermomucor indicae-seudaticae N31 (Milk-clotting protease from the newly isolated Thermomucor indicae-seudaticae N31)

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

Ligninolytic activity from newly isolated basidiomycete strains and effect of these enzymes on the azo dye orange II decolourisation

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

Endoglucanase production with the newly isolated Myceliophtora sp. i-1d3b in a packed bed solid state fermentor

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

Optimization of polysaccharides production by bacteria isolated from soil

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

Crystallization of bothrombin, a fibrinogen-converting serine protease isolated from the venom of Bothrops jararaca

Bothrombin, a snake-venom serine protease, specifically cleaves fibrinogen, releasing fibrinopeptide A to form non-crosslinked soft clots, aggregates platelets in the presence of exogeneous fibrinogen and activates blood coagulation factor VIII. Bothrombin shares high sequence homology with other snake-venom proteases such as batroxobin (94% identity), but only 30 and 34% identity with human alpha-thrombin and trypsin, respectively. Single crystals of bothrombin have been obtained and X-ray diffraction data have been collected at the Laboratorio Nacional de Luz Sincrotron to a resolution of 2.8 Angstrom. The crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 94.81, b = 115.68, c = 155.97 Angstrom.

Purification, characterization and crystallization of Jararacussin-I, a fibrinogen-clotting enzyme isolated from the venom of Bothrops jararacussu

A fibrinogen-clotting enzyme, Jararacussin-I, was purified from the venom of Bothrops jararacussu by a combination of ion exchange chromatography using Resource 15S resin and affinity chromatography using Benzamidine Sepharose 6B resin. Jararacussin-I displays a molecular mass of 28 kDa as estimated by sodium dodecyl sulphate-PAGE and possesses an isoetectric point of 5.0. The coagulant specific activity of the enzyme was determined to be 45.8 NIH U/mg using bovine fibrinogen as the substrate and the esterase specific activity was determined to be 258.7 U/mg. The protease inhibitors, benzamidine and DTT inhibited the esterase specific activity by 72.4 and 69.7%, respectively. The optimal temperature and pH for the degradation of both chains of fibrinogen and esterase specific activity were determined to be 37 degreesC and 7.4-8.0, respectively. The enzyme was inactivated at both 4 and 75 T. Single crystals of Jararacussin-I were obtained and complete three-dimensional X-ray diffraction data was collected at the Brazilian National Synchrotron Source (LNLS) to a resolution of 2.4 Angstrom. (C) 2002 Published by Elsevier B.V. Ltd.

Structure of a Lys49 phospholipase A(2) homologue isolated from the venom of Bothrops nummifer (jumping viper)

Lys49-Phospholipase A(2) (Lys49-PLA(2)) homologues damage membranes by a Ca2+-independent mechanism which does not involve catalytic activity, We have solved the structure of myotoxin-I, a Lys49-PLA(2) homologue isolated from the venom of Bothrops nummifer (jumping viper) at 2.4 Angstrom resolution using molecular replacement techniques. The final model has been refined to a final R-factor of 18.4% (R-free = 23.2%), and shows excellent geometry, the myotoxin-I from Bothrops nummifer is dimeric in the crystalline state as has been observed for other Lys49-PLA(2) homologues. In addition, a continuous electron density in the active site and substrate binding channel could be successfully modeled as a fatty-acid molecule. (C) 1999 Elsevier B.V. Ltd, All rights reserved.

Crystallization and preliminary X-ray crystallographic studies of Protac (R), a commercial protein C activator isolated from Agkistrodon contortrix contortrix venom

The protein C pathway plays an important role in the control and regulation of the blood coagulation cascade and prevents the propagation of the clotting process on the endothelium surface. In physiological systems, protein C activation is catalyzed by thrombin, which requires thrombomodulin as a cofactor. The protein C activator from Agkistrodon contortrix contortrix acts directly on the zymogen of protein C converting it into the active form, independently of thrombomodulin. Suitable crystals of the protein C activator from Agkistrodon contortrix contortrix were obtained from a solution containing 2 M ammonium sulfate as the precipitant and these crystals diffracted to 1.95 angstrom resolution at a synchrotron beamline. The crystalline array belongs to the monoclinic space group C2 with unit cell dimensions a=80.4, b = 63.3 and c = 48.2 angstrom, alpha = gamma = 90.0 degrees and beta = 90.8 degrees. (C) 2005 Elsevier B.V. All rights reserved.

Crystallization and preliminary X-ray crystallographic analysis of the heterodimeric crotoxin complex and the isolated subunits crotapotin and phospholipase A(2)

Crotoxin, a potent neurotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus, exists as a heterodimer formed between a phospholipase A(2) and a catalytically inactive acidic phospholipase A(2) analogue (crotapotin). Large single crystals of the crotoxin complex and of the isolated subunits have been obtained. The crotoxin complex crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 38.2, b = 68.7, c = 84.2 angstrom, and diffracted to 1.75 angstrom resolution. The crystal of the phospholipase A(2) domain belongs to the hexagonal space group P6(1)22 (or its enantiomorph P6(5)22), with unit-cell parameters a = b = 38.7, c = 286.7 angstrom, and diffracted to 2.6 angstrom resolution. The crotapotin crystal diffracted to 2.3 angstrom resolution; however, the highly diffuse diffraction pattern did not permit unambiguous assignment of the unit-cell parameters.

Antimicrobial activity of an L-amino acid oxidase isolated from Bothrops leucurus snake venom

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Purification, biochemical and functional characterization of miliin, a new thiol-dependent serine protease isolated from the latex of Euphorbia milii

Miliin, a new thiol-dependent serine protease purified from the latex of Euphorbia milii possesses a molecular weight of 79 kDa, an isoelectric point of 4.3 and is optimally active at 60 degrees C in the pH range of and 7.5-11.0. Activity tests indicate that milliin is a thiol-dependent serine protease.

A new lipase isolated from oleaginous seeds from Pachira aquatica (Bombacaceae)

A new lipase from seeds of Pachira aquatica was purified to homogeneity by SDS-PAGE obtaining an enzyme with a molecular weight of approximately 55 kDa. The purified lipase exhibited maximum activity at 40 degrees C and pH 8.0, for an incubation time of 90 min. Concerning temperature stability, at the range from 4 to 50 degrees C, it retained approximately 47% of its original activity for 3 h. The enzyme activity increased in the presence of Ca(++) and Mg(++), but was inhibited by Hg(++), Mn(++), Zn(++), Al(+++) and various oxidizing and reducing agents. The lipase was highly stable in the presence of organic solvents, and its activity was stimulated by methanol. The values of K(m) and V(max) were 1.65 mM and 37.3 mu mol mL(-1) min(-1), respectively, using p-nitrophenylacetate as substrate. The enzyme showed preference for esters of long-chain fatty acids, but demonstrated significant activity against a wide range of substrates.

Preliminary functional characterization, cloning and primary sequence of Fastuosain, a cysteine peptidase isolated from fruits of Bromelia fastuosa

The present work reports the characterization of Fastuosain, a novel cysteine protease of 25kDa, purified from the unripe fruits of Bromelia fastuosa, a wild South American Bromeliaceae. Proteolytic activity, measured using casein and synthetic substrates, was dependent on the presence of thiol reagents, having maximum activity at pH 7.0. The present work reports cDNA cloning of Fastuosain; cDNA was amplified by PCR using specific primers. The product was 1096pb long. Mature fastuosain has 217 residues, and with the proregion has a total length of 324 residues. Its primary sequence showed high homology with ananain(74%), stem bromelain (66%) and papain (44%).

Three exopolysaccharides of the beta-(1 -> 6)-D-glucan type and a beta-(1 -> 3;1 -> 6)-D-glucan produced by strains of Botryosphaeria rhodina isolated from rotting tropical fruit

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

Subcellular localization of the coat protein in tobacco cells infected by cucumber mosaic virus isolated from Catharanthus roseus

Electron microscopy and immunolabelling with antiserum specific to cucumber mosaic virus coat protein were used to examine tobacco leaf cells infected by cucumber mosaic virus isolated from Catharanthus roseus (CMV-Cr). Crystalline and amorphous inclusions in the vacuoles were the most obvious cytological modifications seen. Immunogold labelling indicated that the crystalline inclusion was made up of virus particles and amorphous inclusions contained coat protein. Rows of CMV-Cr particles were found between membranes of dictyosomes, but membranous bodies and tonoplast-associated vesicles were not evident. Virus particles and/or free coat protein were easily detected in the cytoplasm by immunolabelling. No gold labelling was found within nuclei, chloroplasts and mitochondria.