Effect of surfactants and polyethylene glycol on the activity and stability of a lipase from oilseeds of Pachira aquatica

Lipases from oilseeds have a great potential for commercial exploration as industrial enzymes. Lipases are used mixed with surfactants in cleaning and other formulated products, and accordingly, both components must be compatible with each other. This work presents the results of the effects of anionic, cationic and nonionic surfactants, polyethylene glycol and urea on the activity and stability of a lipase extracted of oilseeds from Pachira aquatica. The enzyme was purified and the spectrophotometric assays were done using p-nitrophenyl acetate (p-NPA) as substrate pH 7.5 and 25 degrees C. The activity was significantly enhanced by the cationic surfactant CTAB. Bile salts increased the lipase activity in the tested concentration range, whereas anionic and nonionic surfactants showed an inhibitory effect. Aqueous solutions of PEG activated the lipase and maximum activation (161%) occurred in PEG 12,000. This effect on lipase that can be due to exposition of some hydrophobic residues located in the vicinity of the active site or aggregation.

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.

Glyceraldehyde-3-phosphate dehydrogenase of Paracoccidioides brasiliensis is a cell surface protein involved in fungal adhesion to extracellular matrix proteins and interaction with cells

The pathogenic fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a pulmonary mycosis acquired by inhalation of fungal airborne propagules, which may disseminate to several organs and tissues, leading to a severe form of the disease. Adhesion to and invasion of host cells are essential steps involved in the infection and dissemination of pathogens. Furthermore, pathogens use their surface molecules to bind to host extracellular matrix components to establish infection. Here, we report the characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of P. brasiliensis as an adhesin, which can be related to fungus adhesion and invasion. The P. brasiliensis GAPDH was overexpressed in Escherichia coli, and polyclonal antibody against this protein was obtained. By immunoelectron microscopy and Western blot analysis, GAPDH was detected in the cytoplasm and the cell wall of the yeast phase of P. brasiliensis. The recombinant GAPDH was found to bind to fibronectin, laminin, and type I collagen in ligand far-Western blot assays. of special note, the treatment of P. brasiliensis yeast cells with anti-GAPDH polyclonal antibody and the incubation of pneumocytes with the recombinant protein promoted inhibition of adherence and internalization of P. brasiliensis to those in vitro-cultured cells. These observations indicate that the cell wall-associated form of the GAPDH in P. brasiliensis could be involved in mediating binding of fungal cells to fibronectin, type I collagen, and laminin, thus contributing to the adhesion of the microorganism to host tissues and to the dissemination of infection.

Production of extracellular alkaline proteases by Aspergillus clavatus

The production of extracellular alkaline 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 casein pH 9.5 at 37degreesC. The highest alkaline proteolytic activity (38 U/ml) was verified for culture medium containing glucose and casein at 1% (w/v) as substrates, obtained from cultures developed at 25degreesC for 6 days. Cultures developed in Vogel medium with glucose at 2% (w/v) and 0.2% (w/v) NH4NO3 showed higher proteolytic activity (27 U/ml) when compared to the cultures with 1% of the same sugar. Optimum temperature was 40degreesC and the half-lives at 40, 45 and 50degreesC were 90, 25 and 18 min, respectively. Optimum pH of enzymatic activity was 9.5 and the enzyme was stable from pH 6.0 to 12.0.

Collagen type VI is a component of the extracellular matrix microfibril network of the prostatic stroma

Stroma-epithelium relationships are of great relevance in prostatic morphogenesis and physiology, However, little knowledge exists about either stromal cells or extracellular matrix composition and arrangement in this system, Ultrastructural analysis revealed the existence of a microfibrillar system which occupies large areas of the rat prostatic stroma, In this work, we have applied immunocytochemistry and an ATP treatment for the ultrastructural identification of collagen type VI microfibrils, aiming at examining its participation in the prostatic microfibrillar network. Immunocytochemistry was also extended to a human case of prostatic nodular hyperplasia, Both methods succeeded in identifying collagen type VI in the rat ventral prostate, Collagen type VI is evenly distributed throughout the stroma but mainly associated with the basal lamina, collagen fibrils, and around the stromal cells, the use of ATP treatment allowed for the discrimination between collagen type VI and elastin-associated microfibrils, and demonstrated that these two classes of microfibrils establish an extended, mixed, and open network. The same aspects of association with the basal lamina, with stromal cells (particularly with smooth muscle cells), and with fibrillar components of the stroma were observed in the human tissue, We suggest that the collagen type VI and elastin-associated microfibril system may be involved in the control of some aspects of cellular behavior and may also play a structural role, maintaining the organ integrity after the deformations occurring under smooth muscle contraction.

Extracellular matrix of porcine pericardium: Biochemistry and collagen architecture

Pericardial tissue has been used to construct bioprostheses employed in the repair of different kinds of injuries, mostly cardiac. However, calcification and mechanical failure have been the main causes of the limited durability of cardiac bioprostheses constructed with bovine pericardium. In the course of this work, a study was conducted on porcine fibrous pericardium, its microscopic structure and biochemical nature. The general morphology and architecture of collagen were studied under conventional light and polarized light microscopy. The biochemical study of the pericardial matrix was conducted according to the following procedures: swelling test, hydroxyproline and collagen dosage, quantification of amino acids in soluble collagen, component extraction of the extracellular matrix of the right and left ventral regions of pericardium with different molarities of guanidine chloride, protein and glycosaminoglycan (GAG) dosage, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and total GAG analysis. Microscopic analysis showed collagen fibers arranged in multidirectionally oriented layers forming a closely knit web, with a larger number of fibers obliquely oriented, initiating at the lower central region toward the upper left lateral relative to the heart. No qualitative differences were found between proteins extracted from the right and left regions. Likewise, no differences were found between fresh and frozen material. Protein dosages from left frontal and right frontal pericardium regions showed no significant differences. The quantities of extracted GAGs were too small for detection by the method used. Enzymatic digestion and electrophoretic analysis showed that the GAG found is possibly dermatan sulfate. The proteoglycan showed a running standard very similar to the small proteoglycan decorin.

Protease activity in extracellular products secreted in vitro by trophozoites of Giardia duodenalis

There are evidences that Giardia trophozoites contain and/or release proteolytic enzymes that may be implicated in pathogenesis of giardiasis. This report describes a preliminary characterization of the proteolytic activity in excretory/secretory (E/S) products of Giardia duodenalis trophozoites of an axenic Brazilian strain (BTU-11) and the reference strain Portland 1 (P1). The protease activity of E/S products in conditioned medium by trophozoites of each strain was analyzed using substrate (gelatin and collagen) impregnated SDS-PAGE and hemoglobin assay. The protease characterization was based on inhibition assays including synthetic inhibitors. Proteolytic products were detected in the conditioned medium by trophozoites of both assayed strains. In the gels containing copolymerized gelatin and collagen, E/S products promoted degradation of the substrates and the most evident proteolysis zones were distributed in the migration regions of 77 to 18 kDa and 145 to 18 kDa, respectively, in the patterns of gelatinolytic and collagenolytic activities. Degradation of hemoglobin was also observed, and the pattern of hydrolysis was similar in both E/S products assayed. Inhibition assays showed that the main proteolytic activity in both E/S products is due to cysteine proteases although the presence of serine proteases was also indicated, mainly in the hydrolysis of hemoglobin.

Characterization of a thermostable extracellular tannase produced under submerged fermentation by Aspergillus ochraceus

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

Porphyromonas gingivalis-mediated shedding of extracellular matrix metalloproteinase inducer (EMMPRIN) by oral epithelial cells: a potential role in inflammatory periodontal disease

Extracellular matrix metalloproteinase inducer (EMMPRIN) or CD 147 is a transmembrane glycoprotein expressed by various cell types, including oral epithelial cells. Recent studies have brought evidence that EMMPRIN plays a role in periodontitis. In the present study, we investigated the effect of Porphyromonas gingivalis, a major pathogen in chronic periodontitis, on the shedding of membrane-anchored EMMPRIN and on the expression of the EMMPRIN gene by oral epithelial cells. A potential contribution of shed EMMPRIN to the inflammatory process of periodontitis was analyzed by evaluating the effect of recombinant EMMPRIN on cytokine and matrix metalloproteinase (MMP) secretion by human gingival fibroblasts. ELISA and immunofluorescence analyses revealed that P. gingivalis mediated the shedding of epithelial cell-surface EMMPRIN in a dose- and time-dependent manner. Cysteine proteinase (gingipain)-deficient P. gingivalis mutants were used to demonstrate that both Arg- and Lys-gingipain activities are involved in EMMPRIN shedding. Real-time PCR showed that P. gingivalis had no significant effect on the expression of the EMMPRIN gene in epithelial cells. Recombinant EMMPRIN induced the secretion of IL-6 and MMP-3 by gingival fibroblasts, a phenomenon that appears to involve mitogen activated protein kinases. The present study brought to light a new mechanism by which P. gingivalis can promote the inflammatory response during periodontitis. (C) 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Extracellular tannase from Emericella nidulans showing hypertolerance to temperature and organic solvents

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

On the stability of the extracellular hemoglobin of Glossoscolex paulistus, in two iron oxidation states, in the presence of urea

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