Efecto antibacteriano in vitro de extractos de Caesalpinia spinosa sobre cepas de Staphylococcus aureus resistentes a antibióticos betalactámicos / In vitro antimicrobial effect of Caesalpinia spinosa extracts against betalactam antibiotics resistant Staphylococcus aureus strains

Objetivo. Determinar el efecto antimicrobiano de extractos etanólicos de Caesalpinia spinosa sobre cepas de Staphylococcus aureus penicilino y meticilino resistentes. Métodos. Se utilizaron las cepas de S. aureus ATCC 11632 y 33592, los extractos etanólicos de hojas, vainas y semillas de C. spinosa, se obtuvieron por maceración en concentraciones de 25%, 50%, 75% y 100%. Se utilizó el método de Kirby-Bauer, los discos de papel filtro se cargaron con los extractos y se depositaron sobre el medio, inoculado con una suspensión de S. aureus a 0,5 McFarland. El control positivo fueron discos de ampicilina y el control negativo discos impregnados con etanol. Después de 24 horas se midieron los diámetros de los halos con un calibrador Vernier. Resultados. Se registraron halos de hasta 18 mm de diámetro con los extractos de hojas al 100%, 17 mm con extractos de vainas y 14 mm con extractos de semillas sobre S. aureus ATCC 33592. En el caso de la cepa ATCC 11632, se registraron halos de hasta 14 mm con extractos de hojas y vainas al 100%, y de hasta 8 mm con extractos de semillas. La prueba de ANOVA indicó que existieron diferencias significativas entre los halos obtenidos con los diferentes tipos de extractos, a diferentes concentraciones. Conclusión. Se determinó que todos los extrac- tos de C. spinosa poseen actividad antimicrobiana sobre las dos cepas estudiadas, con un patrón directamente proporcional entre el efecto y la concentración.

Objective. To determine the antimicrobial effect of ethanolic extracts from Caesalpinia spinosa against penicillin and methicillin resistant strains of Staphylococcus aureus penicillin and methicillin. Methods. S. aureus ATCC 11632 and 33592 strains were used, ethanolic extracts from C. spinosa leaves and pods were obtained by maceration at concentrations of 25%, 50%, 75% and 100%. The Kirby-Bauer method was used, where filter paper discs were loaded with leaf, pod and seed extracts and deposited on the medium, inoculated with a 0.5 McFarland suspension of S. aureus. Ampicillin discs were used as positive con- trol and ethanol-impregnated discs as negative control. After 24 hours, the diameters of the halos were measured with a Vernier caliper. Results. Haloes up to 18 mm in diameter with 100% leaf extracts, 17 mm with pod extracts and 14 mm with seed extracts were recorded against S. aureus ATCC 33592; for ATCC strain 11632, haloes up to 14 mm with 100% leaf and pod extracts and up to 8 mm with seed extracts were recorded. The ANOVA test indicated significant differences between the inhibition halos obtained with the different types of extracts, at different concentrations. Conclusion. It was determined that all C. spinosa extracts possess antimicrobial activity against the two strains studied, with a pattern directly proportional between the effect and concentration.

Cloning, heterologous expression and properties of a recombinant active turnip peroxidase.

Turnip (Brassica napus) roots peroxidase isoforms have been used in diagnostic kits and can also efficiently polymerize phenolic compounds from wastewaters. Heterologous expression of a turnip acidic peroxidase (BnPA) was investigated to increase availability of this widely used enzyme. The mature BnPA was ligated into the pET28a(+) vector and used to transform Escherichia coli Rosetta 2. Recombinant BnPA peroxidase was overexpressed and accumulated in inclusion bodies from which it was purified to homogeneity by immobilized metal affinity chromatography under denaturing conditions. Peroxidase activity was observed after a refolding process under oxidative conditions. The yield of pure recombinant BnPA was 29 mg L(-1) of culture with a specific activity of 981 ± 20 ABTS units mg(-1) at optimal conditions (pH 6, 45 °C). Recombinant BnPA showed similar kinetic properties compared to native turnip peroxidase, and its secondary structure evaluated by circular dichroism comprised 20% α-helix, 32% ß-sheet and 48% random structure. Recombinant BnPA showed high yield and good kinetic properties which are key steps for future structure-function studies and biotechnological applications.

Homologue expression of a ß-xylosidase from native Aspergillus niger.

Xylan constitutes the second most abundant source of renewable organic carbon on earth and is located in the cell walls of hardwood and softwood plants in the form of hemicellulose. Based on its availability, there is a growing interest in production of xylanolytic enzymes for industrial applications. ß-1,4-xylan xylosidase (EC 3.2.1.37) hydrolyses from the nonreducing end of xylooligosaccharides arising from endo-1,4-ß-xylanase activity. This work reports the partial characterization of a purified ß-xylosidase from the native strain Aspergillus niger GS1 expressed by means of a fungal system. A gene encoding ß-xylosidase, xlnD, was successfully cloned from a native A. niger GS1 strain. The recombinant enzyme was expressed in A. niger AB4.1 under control of A. nidulans gpdA promoter and trpC terminator. ß-xylosidase was purified by affinity chromatography, with an apparent molecular weight of 90 kDa, and showed a maximum activity of 4,280 U mg protein(-1) at 70°C, pH 3.6. Half-life was 74 min at 70°C, activation energy was 58.9 kJ mol(-1), and at 50°C optimum stability was shown at pH 4.0-5.0. ß-xylosidase kept residual activity >83% in the presence of dithiothreitol (DTT), ß-mercaptoethanol, sodium dodecyl sulfate (SDS), ethylenediaminetetraacetate (EDTA), and Zn(2+). Production of a hemicellulolytic free xylosidase showed some advantages in applications, such as animal feed, enzymatic synthesis, and the fruit-juice industry where the presence of certain compounds, high temperatures, and acid media is unavoidable in the juice-making process.

A putative peroxidase cDNA from turnip and analysis of the encoded protein sequence.

A putative peroxidase cDNA was isolated from turnip roots (Brassica napus L. var. purple top white globe) by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Total RNA extracted from mature turnip roots was used as a template for RT-PCR, using a degenerated primer designed to amplify the highly conserved distal motif of plant peroxidases. The resulting partial sequence was used to design the rest of the specific primers for 5' and 3' RACE. Two cDNA fragments were purified, sequenced, and aligned with the partial sequence from RT-PCR, and a complete overlapping sequence was obtained and labeled as BbPA (Genbank Accession No. AY423440, named as podC). The full length cDNA is 1167bp long and contains a 1077bp open reading frame (ORF) encoding a 358 deduced amino acid peroxidase polypeptide. The putative peroxidase (BnPA) showed a calculated Mr of 34kDa, and isoelectric point (pI) of 4.5, with no significant identity with other reported turnip peroxidases. Sequence alignment showed that only three peroxidases have a significant identity with BnPA namely AtP29a (84%), and AtPA2 (81%) from Arabidopsis thaliana, and HRPA2 (82%) from horseradish (Armoracia rusticana). Work is in progress to clone this gene into an adequate host to study the specific role and possible biotechnological applications of this alternative peroxidase source.

Chemical modification of turnip peroxidase with methoxypolyethylene glycol enhances activity and stability for phenol removal using the immobilized enzyme.

Peroxidase from turnip roots (TP) was isolated followed by modification with methoxypolyethylene glycol (MPEG). The catalytic activity of the modified TP (MTP) on ABTS increased 2.5 times after 80 min of reaction. MTP showed a KM similar value to that of TP, but a significantly greater kcat for ABTS oxidation, in aqueous buffer. Chemical modification produced an enhanced stability in organic solvents and increased thermal stability of about 4 times that of TP, in aqueous buffer at 70 degrees C. Circular dichroism showed that MPEG modification decreased TP alpha-helical structure from 26 to 16% and increased beta-turns from 26 to 34%, resulting in an enhanced conformational stability. The temperature at the midpoint of thermal denaturation (melting temperature) increased from 57 to 63 degrees C after modification. MTP was immobilized in alginate beads (IMTP) and tested for oxidative polymerization of concentrated phenolic synthetic solutions, achieving 17 effective contact cycles removing >65% phenols. IMTP may be useful for the development of an enzymatic process for wastewater effluent treatment.

Polyethylene glycol improves phenol removal by immobilized turnip peroxidase.

Purified peroxidase from turnip (Brassica napus L. var. esculenta D.C.) was immobilized by entrapment in spheres of calcium alginate and by covalent binding to Affi-Gel 10. Both immobilized Turnip peroxidase (TP) preparations were assayed for the detoxification of a synthetic phenolic solution and a real wastewater effluent from a local paints factory. The effectiveness of phenolic compounds (PC's) removal by oxidative polymerization was evaluated using batch and recycling processes, and in the presence and in the absence of polyethylene glycol (PEG). The presence of PEG enhances the operative TP stability. In addition, reaction times were reduced from 3h to 10 min, and more effective phenol removals were achieved when PEG was added. TP was able to perform 15 reaction cycles with a real industrial effluent showing PC's removals >90% PC's during the first 10 reaction cycles. High PC's removal efficiencies (>95%) were obtained using both immobilized preparations at PC's concentrations <1.2mM. Higher PC's concentrations decreased the removal efficiency to 90% with both preparations after the first reaction cycle, probably due to substrate inhibition. On the other hand, immobilized TP showed increased thermal stability when compared with free TP. A large-scale enzymatic process for industrial effluent treatment is expected to be developed with immobilized TP that could be stable enough to make the process economically feasible.

Food-associated lactic acid bacteria with antimicrobial potential from traditional Mexican foods.

This work was conducted to identify indigenous LAB capable of antimicrobial activity, present in traditional Mexican-foods with potential as natural preservatives. A total of 27 artisan unlabeled Mexican products were evaluated, from which 94 LAB strains were isolated, and only 25 strains showed antimicrobial activity against at least one pathogen indicator microorganism. Most of the inhibitory activity showed by the isolated LAB strains was attributed to pH reduction by organic acids. Lactobacillus and Lactococcus strains were good acid producers, depending on the substrate, and may enhance the safety of food products. Cell free cultures of Leuconostoc mesenteroides CH210, and PT8 (from chorizo and pulque, respectively) reduced the number of viable cells of enteropathogenic E. coli in broth system. Lb. plantarum CC10 (from "madre" of vinegar) showed significant inhibitory effect against S. aureus 8943. E. faecium QPII (from panela cheese) produced a bacteriocin with wide anti-L. monocytogenes activity. Selected LAB from traditional Mexican foods showed good potential as bio-preservatives.

Anti-Listeria monocytogenes bacteriocin-like inhibitory substances from Enterococcus faecium UQ31 isolated from artisan Mexican-style cheese.

Artisan fresh Mexican-style cheeses are commonly made from raw milk that provides not only rich flavors, but also a diversity of associated lactic acid bacteria (LAB) strains. Enterococcus faecium UQ31 was isolated from panela cheese and produced bacteriocin-like inhibitory substances (BLIS) with a strong anti-Listeria activity. A modified pH-mediated adsorption-desorption purification process resulted in (after SDS-PAGE) two bands showing antimicrobial activities, where most of the activity corresponded to the band with an estimated molecular weight of 7.5 kDa. The BLIS produced by E. faecium UQ31 were heat resistant, stable at ambient storage conditions, and active in the pH range 5--9. The BLIS antimicrobial activities were detected during logarithmic growth phase and remained constant until the end of incubation time (19 h). These BLIS showed a wide anti-Listeria monocytogenes spectra. The E. faecium UQ31 strain or their BLIS represent a promising potential as antimicrobial food preservatives.

Purification and properties of a neutral peroxidase isozyme from turnip (Brassica napus L. Var. Purple Top White Globe) roots.

A neutral peroxidase isozyme (pI 7.2) from turnip roots (TNP) was purified to homogeneity and partially characterized. TNP is a monomeric glycoprotein with 9.1% carbohydrate content and a molecular weight of 36 kDa. Optimum pH values for activity using 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and guaiacol as H donors were 4.5 and 5.5, whereas the K(m) values were 0.7 and 3.7 mM, respectively. The ABTS K(m) was approximately 7 times higher than that reported for basic commercial horseradish peroxidase (HRP-C). TNP retained approximately 70% activity after 11 min of heating at 65 degrees C, whereas the activation energy for inactivation (132 kJ/mol) was higher than or comparable to that of other peroxidases. The low ABTS K(m) and high specific activity (1930 units/mg) gave a high catalytic efficiency (500 M(-1) s(-1)). These properties make TNP an enzyme with a high potential as an alternative to HRP in various applications.

Purification and partial characterization of three turnip (Brassicanapus L. var. esculenta D.C.) peroxidases.

Three turnip peroxidases (fractions C1, C2, and C3) were partially purified and characterized, to permit study of their feasibility for use in clinical and enzyme immunoassays. These fractions represented 20% of the initial activity, and fractions C1 and C2 were purified to homogeneity. The optimum pH was between 5.0 and 5.5, while optimum temperature ranged from 40 to 55 degrees C. The ABTS K(m) values for the two acidic fractions (C2 and C3) were 0.70 and 0.42 mM, respectively; about 5 times lower than that reported for the acidic commercial horseradish peroxidase (HRP). Fraction C3 had 4 times higher K(m) value than commercial cationic HRP. The molecular weights determined by SDS-PAGE ranged from 39.2 to 42.5 kDa. Activation energies for inactivation were 113 (C1), 130 (C2), and 172 kJ/mol (C3) which are higher or comparable to other peroxidase isoenzymes reported. Fractions C1 and C3 represent an alternative source of peroxidase because of their higher purification yield and specific activity, when compared to fraction C2.

Aggregation and collapse in a mechanical model of fungal tip growth.

Interconnected hyphal tubes form the mycelia of a fungal colony. The growth of the colony results from the elongation and branching of these single hyphae. The material being incorporated into the extending hyphal wall is supplied by vesicles which are formed further back in the hyphal tip. Such wall-destined vesicles appear conspicuously concentrated in the interior of the hypha, just before the hyphal apex, in the form of an apical body or Spitzenkörper. The cytoskeleton of the hyphal tube has been implicated in the organisation of the Spitzenkörper and the transport of vesicles, but as yet there is no postulated mechanism for this. We propose a mechanism by which forces generated by the cytoskeleton are responsible for biasing the movement of vesicles. A mathematical model is derived where the cytoskeleton is described as a viscoelastic fluid. Viscoelastic forces are coupled to the conservation equation governing the vesicle dynamics, by weighting the diffusion of vesicles via the strain tensor. The model displays collapse and aggregation patterns in one and two dimensions. These are interpreted in terms of the formation of the Spitzenkörper and the initiation of apical branching.

Non-linear optimization of biotechnological processes by stochastic algorithms: application to the maximization of the production rate of ethanol, glycerol and carbohydrates by Saccharomyces cerevisiae.

A non-linear optimization, based on an stochastic multi-start search algorithm, has been applied to the maximization of the production rates of ethanol, glycerol and carbohydrates by Saccharomyces cerevisiae. This optimization is applied to two alternative (non-linear) model representations of the same system, namely the Michaelis-Menten and the generalized mass action forms. We find a complete agreement between the results obtained using both representations. This is, maximization of the ethanol production rate requires modulation of up to six enzymes, while modification of only one enzyme is sufficient to obtain a significant improvement in the production rate of glycerol and carbohydrates. When the results are compared with those previously obtained using an indirect linear optimization method (Torres, N.V., Voit, E.O., González-Alcón, C., Rodríguez, F. 1997. An integrated optimization method for biochemical systems. Description of method and application to ethanol, glycerol and carbohydrate production in S. cerevisiae. Biotechnol. Bioeng. 55(5), 758-772.), we find close agreement between both optimization techniques. Qualitatively, both optimization approaches render the same profile of enzymes to be modulated, while quantitatively, discrepancies arise when the objective function is the maximization of the ethanol production rate. Reasons for such discrepancies and an evaluation of the advantages of each method (linear vs non-linear) are given.

Studies on the purification of peroxidase from horseradish roots using reverse micelles.

Horseradish peroxidase (HRP) was successfully purified from horseradish roots by a two-stage reverse-micellar extraction from the dialyzed aqueous extract. The anionic surfactant AOT dissolved in isooctane was used to produce the reverse-micellar phases. The narrow pH range at which HRP solubilization occurred was exploited to remove most of the contaminant proteins in the first forward extraction. In the second extraction stage, HRP was selectively solubilized and concentrated by using a volume ratio of 10 between the aqueous and organic phases. The HRP final specific activity was 86 guaiacol U mg-1, obtained with a purification factor of 80 and yield of 46%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two overlapping bands, with HRP corresponding to that at 43.8 kDa. Image analysis on isoelectric focusing (IEF) gels showed that the HRP was 80% pure. Ion exchange liquid chromatography showed that most of the specific activity was due to the basic isoenzyme with pI 8.5, which comprises 33.5% of the product. There were high HRP losses as a precipitate at the interface when direct reverse-micellar extraction was attempted from the crude extract. It is believed that the hydrophobic environment near the haem group of the HRP basic isoenzyme favors complex formation with the surfactant, and that this is promoted at higher protein concentrations.

Protein extraction by reverse micelles: studies on the recovery of horseradish peroxidase.

Phase transfer studies were carried out on the solubilization of horseradish peroxidase (HRP) (E.C. 1.11.1.7) in reverse micelles formed in isooctane using the anionic surfactant, aerosol OT, at concentrations between 50 and 110 mM. The selectivity of this methodology was tested, because the HRP used comprised a mixture of seven different isoenzymes with a wide range of isoelectric points. Forward and backward transfers were carried out in wellstirred vessels until equilibrium was reached. Significant protein partitioning could only be obtained by using NaCl to adjust ionic strength in pH range between 1.5 and 3.5, with a maximum at pH 3. The back transfer process was best at pH 8 with 80 mM phosphate buffer and 1 M KCI. A loss of 1% to 3% of the surfactant through precipitation at the interface at pH<4 was observed, which may be due to instability in this pH region, because, even without protein, a similar precipitate was noticed. Protein partitioning was approximately constant when the ionic strength was increased up to 1 MNaCl at pH 3, but protein recovery in back transfer decreased accordingly. Hydrophobic interactions together with association between the protein and surfactant might be responsible for that behavior. Protein partitioning remained the same when the surfactant concentration was decreased to 50 mM, at the expense of higher variability. HPLC chromatograms showed no apparent damage to the protein after reverse micellar extraction. Protein partitioning is best when the temperature is kept at 25 degrees C. The amount of protein and specific activity recovered strongly depends on the phase ratio used during forward transfer. Overall activity recovery varied from 87% to 136% when the phase ratio was increased from 1:1 to 30:1 in forward transfer. This behavior may be due to a change in the ratio of the three isoenzymes recovered after the backward transfer process, with the most active one being increasingly enriched at higher phase ratios.

Hemodynamic correlates of vectorcardiographic QRS loops in pure mitral stenosis.

The hemodynamic correlates of the vectorcardiographic types of right ventricular hypertrophy (RVH) according to Chou and Helm and those with normal QRS loop in the horizontal plane of Frank system were analyzed in 100 patients with pure mitral stenosis. All underwent right and left heart catheterization. Additionally, coronary arteriography was done on 16 whose ages were above 40. Type A RVH was associated with the most severe hemodynamic alterations with markedly elevated total pulmonary vascular resistance (TPVR), mean pulmonary artery pressure (MPAP), peak right ventricular pressure (RRVP) and the smallest mitral valve area (MVA). The severity of these parameters were to a lesser degree obtainable in type C but with no significant difference from type A (p greater than 0.05). However, types A and C were clearly separated from type B and normal QRS loop (p less than 0.05). Type B RVH and normal QRS loop showed milder hemodynamic changes and were not significantly different (p greater than 0.05). Our results indicate that in pure mitral stenosis the development of RVH is from a normal loop into type B, C and A reflecting an increasing severity of hemodynamic changes which affect the right ventricle. This order of development is different from the traditional view.