Acylase enzymes disrupting quorum sensing alter the transcriptome and phenotype of Pseudomonas aeruginosa, and the composition of bacterial biofilms from wastewater treatment plants.

Biofilms represent an essential way of life and colonization of new environments for microorganisms. This feature is regulated by quorum sensing (QS), a microbial communication system based on autoinducer molecules, such as N-acyl-homoserine lactones (AHLs) in Gram negative bacteria. In artificial ecosystems, like Wastewater Treatment Plants (WWTPs), biofilm attachment in filtration membranes produces biofouling. In this environment, the microbial communities are mostly composed of Gram-negative phyla. Thus, we used two AHLs-degrading enzymes, obtained from Actinoplanes utahensis (namely AuAAC and AuAHLA) to determine the effects of degradation of QS signals in the biofilm formation, among other virulence factors, of a Pseudomonas aeruginosa strain isolated from a WWTP, assessing molecular mechanisms through transcriptomics. Besides, we studied the possible effects on community composition in biofilms from activated sludge samples. Although the studied enzymes only degraded the AHLs involved in one of the four QS systems of P. aeruginosa, these activities produced the deregulation of the complete QS network. In fact, AuAAC -the enzyme with higher catalytic efficiency- deregulated all the four QS systems. However, both enzymes reduced the biofilm formation and pyocyanin and protease production. The transcriptomic response of P. aeruginosa affected QS related genes, moreover, transcriptomic response to AuAAC affected mainly to QS related genes. Regarding community composition of biofilms, as expected, the abundance of Gram-negative phyla was significantly decreased after enzymatic treatment. These results support the potential use of such AHLs-degrading enzymes as a method to reduce biofilm formation in WWTP membranes and ameliorate bacterial virulence.

New insights on nucleoside 2'-deoxyribosyltransferases: a versatile biocatalyst for one-pot one-step synthesis of nucleoside analogs.

In recent years, glycosiltransferases have arisen as standard biocatalysts for the enzymatic synthesis of a wide variety of natural and non-natural nucleosides. Such enzymatic synthesis of nucleoside analogs catalyzed by nucleoside phosphorylases and 2'-deoxyribosyltransferases (NDTs) has demonstrated to be an efficient alternative to the traditional multistep chemical methods, since chemical glycosylation reactions include several protection-deprotection steps. This minireview exhaustively covers literature reports on this topic with the final aim of presenting NDTs as an efficient option to nucleoside phosphorylases for the synthesis of natural and non-natural nucleosides. Detailed comments about structure and catalytic mechanism of described NDTs, as well as their possible biological role, substrate specificity, and advances in detection of new enzyme specificities towards different non-natural nucleoside synthesis are included. In addition, optimization of enzymatic transglycosylation reactions and their application in the synthesis of natural and non-natural nucleosides have been described. Finally, immobilization of NDTs is shown as a practical procedure which leads to the preparation of very interesting biocatalysts applicable to industrial nucleoside synthesis.

Biotechnological applications of penicillin acylases: state-of-the-art.

This review describes the most recent developments in the biotechnological applications of penicillin acylases. This group of enzymes is involved mainly in the industrial production of 6-aminopenicillanic acid and the synthesis of semisynthetic beta-lactam antibiotics. In addition, penicillin acylases can also be employed in other useful biotransformations, such as peptide synthesis and the resolution of racemic mixtures of chiral compounds. Particular emphasis is placed on advances in detection of new enzyme specificities towards other natural penicillins, enzyme immobilization, and optimization of enzyme-catalyzed hydrolysis and synthesis in the presence of organic solvents.

Optimization of 6-aminopenicillanic acid (6-APA) production by using a new immobilized penicillin acylase.

A new immobilized penicillin acylase (ECPVA) was obtained by covalent binding of penicillin acylase from Streptomyces lavendulae on Eupergit C. Enzymic hydrolysis of penicillin V catalysed by ECPVA was optimized using a 2(3) factorial design of experiments, and the selected parameters for this study were pH, temperature and substrate concentration. The immobilized enzyme showed an optimal pH value of 9.5-10.5, and an optimal temperature of 60 degrees C, whereas its soluble counterpart showed the same optimal pH value and a lower optimal temperature of 50 degrees C.

[Reform of health systems in Latin America and the Caribbean: situation and trends]. / Reforma de los sistemas de salud en América Latina y el Caribe: situación y tendencias.

In the early 1990s, most Latin American and Caribbean countries were beginning, or planning to begin, health sector reform processes. This paper presents the status and trends of health sector reform at the end of the 1990s. The authors relied on information in 20 health system and services profiles completed by the Pan American Health Organization between August 1998 and October 1999. The analysis, which follows a methodology that had been applied earlier, was organized on two different levels: (1) monitoring the reform processes (dynamics and content) and (2) evaluating their outcomes. In looking at the dynamics of the reform processes, the article examines the context in which they take place and the actors involved in their different phases: inception, design and negotiation, implementation, and evaluation. The description and analysis of the contents of health sector reform initiatives are organized into 12 broad thematic areas. Outcomes evaluation was only possible in the eight countries that provided enough pertinent information, and should be viewed as preliminary. Nevertheless, the article does present detailed information on the outcomes of health sector reform in terms of equity, effectiveness and quality, efficiency, sustainability, and societal participation and control. The article also discusses the potential causes and determining factors for the observed outcomes.

Effect of hydrogen peroxide on d-amino acid oxidase from Rhodotorula gracilis.

D-amino acid oxidase from Rhodotorula gracilis is a FAD-containing enzyme that belongs to the oxidase class that is characterized by the ability of the reduced flavin to react quickly with oxygen, yielding hydrogen peroxide and the oxidized cofactor. Hydrogen peroxide, necessary for the production of glutaryl-7-ACA from cephalosporin C had a deleterious effect on the enzyme. H(2)O(2) induced the oxidation of tryptophan and cysteine residues of the protein that could be involved in the dimerization process, required for the attainment of a fully competent enzyme. H(2)O(2) had also a kinetic effect on the reaction catalyzed by D-amino acid oxidase. It was a pure noncompetitive inhibitor; the corresponding inhibition constants were K(is) = 0.52 mM and K(ii) = 0.70 mM.

Activation and stabilization of penicillin V acylase from streptomyces lavendulae in the presence of glycerol and glycols.

Penicillin V acylase (EC 3.5.1.11) from Streptomyces lavendulae showed both enhanced activity and stability in mixed water/glycerol and water/glycols solvents. The catalytic activity was increased up to a critical concentration of these cosolvents, but further addition of the latter led to a gradual protein deactivation. The highest stabilizing effect was achieved in the presence of glycerol. Thermal stability was increased proportionally to the concentration of glycerol and glycols in the reaction mixture only if the amount added is below the threshold concentration. Reaction conditions that allow simultaneously enhanced activity and stability in the hydrolysis of penicillin V catalyzed by penicillin V acylase from S. lavendulae could be established.

Prediction of penicillin V acylase stability in water-organic co-solvent monophasic systems as a function of solvent composition.

Hydrolytic activity of penicillin V acylase (EC 3.5.1.11) can be improved by using organic cosolvents in monophasic systems. However, the addition of these solvents may result in loss of stability of the enzyme. The thermal stability of penicillin V acylase from Streptomyces lavendulae in water-organic cosolvent monophasic systems depends on the nature of the organic solvent and its concentration in the media. The threshold solvent concentration (at which half enzymatic activity is displayed) is related to the denaturing capacity of the solvent. We found out linear correlations between the free energy of denaturation at 40 degrees C and the concentration of the solvent in the media. On one hand, those solvents with logP values lower than -1.8 have a protective effect that is enhanced when its concentration is increased in the medium. On the other hand, those solvents with logP values higher than -1.8 have a denaturing effect: the higher this value and concentration, the more deleterious. Deactivation constants of PVA at 40 degrees C can be predicted in any monophasic system containing a water-miscible solvent.

Enhanced production of penicillin V acylase from Streptomyces lavendulae.

A 28 degrees C, Streptomyces lavendulae produced high levels of penicillin V acylase (178 IU/l of culture) when grown on skim milk as the sole nutrient source for 275 h. The enzyme showed catabolite repression by glucose and was produced in the stationary phase of growth. Penicillin V was a good inducer of penicillin V acylase formation, while phenoxyacetic acid, the side-chain moiety of penicillin V, did not alter enzyme production significantly. The enzyme was stable between pH 6 and 11 and at temperatures from 20 degrees C to 55 degrees C. This extracellular enzyme was able to hydrolyse natural penicillins and unable to hydrolyse penicillin G.

Chemical mechanism of D-amino acid oxidase from Rhodotorula gracilis: pH dependence of kinetic parameters.

The variation of kinetic parameters of d-amino acid oxidase from Rhodotorula gracilis with pH was used to gain information about the chemical mechanism of the oxidation of D-amino acids catalysed by this flavoenzyme. d-Alanine was the substrate used. The pH dependence of Vmax and Vmax/Km for alanine as substrate showed that a group with a pK value of 6.26-7.95 (pK1) must be unprotonated and a group with a pK of 10.8-9.90 (pK2) must be protonated for activity. The lower pK value corresponded to a group on the enzyme involved in catalysis and whose protonation state was not important for binding. The higher pK value was assumed to be the amino group of the substrate. Profiles of pKi for D-aspartate as competitive inhibitor showed that binding is prevented when a group on the enzyme with a pK value of 8.4 becomes unprotonated; this basic group was not detected in Vmax/Km profiles suggesting its involvement in binding of the beta-carboxylic group of the inhibitor.

Chemical modification of histidyl residues in D-amino acid oxidase from Rhodotorula gracilis.

D-Amino acid oxidase was inactivated by DEP at 30 degrees C and pH 7.5. The reaction followed pseudo-first-order kinetics with a second-order rate constant of 0.254 mM-1.min-1. The pH dependence of the inactivation showed the involvement of a group with a pK of 6.6. The presence of substrate or benzoate protected the enzyme against inactivation. Difference spectra at 242 nm and the reversal of the inactivation in the presence of 1 M hydroxylamine or 0.1 M NaOH pointed to the modification of histidine residues. The statistical analysis of the residual fractional activity versus the number of modified histidine residues led to the conclusion that one histidine residue is essential for the enzyme activity.

Chemical modification of beta-glucosidase from Trichoderma reesei QM 9414.

The inhibition of beta-glucosidase from Trichoderma reesei QM 9414 by several specific reagents was studied. Diethylpyrocarbonate (DEP) nearly abolished the enzyme activity at concentrations above 10 mM. The presence of substrate or analogs protected the enzyme against inactivation. The reaction followed pseudo-first order kinetics with a second-order rate constant of 0.02 mM-1.min-1. The pH-dependence of the inactivation showed the involvement of a group with a pK of 5.2. Difference spectra at 242 nm and the reversal of the inactivation in the presence of 1 M hydroxylamine indicated the modification of histidine residues. Statistical analysis of residual fractional activity versus the number of modified histidine residues indicated that one histidine residue is essential for catalysis. p-Hydroxymercuribenzoate completely inhibited the enzyme at concentrations of the reagent above 2 mM. Substrate or analogs protected the enzyme against inactivation. The reaction followed pseudo-first order kinetics with a second-order rate constant of 0.002 mM-1.min-1. Treatment of the modified enzyme with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) showed that one cysteine residue was essential for activity. At pH 5.0 2-ethoxy-1-ethoxy-carbonyl-1,2-dihydroquinoline (EEDQ) inactivated the enzyme according to pseudo-first order kinetics with a second-order rate constant of 0.12 min-1. The pH-dependence of the inactivation showed the involvement of a group with a pK of 5.64, indicating the modification of a carboxyl group essential for activity.

Mode of action of endoglucanase III from Trichoderma reesei.

Endoglucanase III (EG III) was purified to homogeneity from the culture medium of Trichoderma reesei QM 9414. It has a molecular mass of 48 kDa, and an isoelectric point of 5.1. Maximal activity was observed between pH4 and 5. Celloligosaccharides and their chromophoric derivatives were used as substrates, and the reaction products were analysed by quantitative h.p.l.c. Nucleophilic competition experiments (between methanol and water) allowed unequivocal assessment of cleavage sites. EG III preferentially released cellobiose (or the corresponding glycoside) from the reducing end of the higher cellodextrins. A putative binding model containing five subsites is proposed. The pH-dependence of 4'-methylumbelliferyl beta-cellotrioside hydrolysis indicates the presence of a protonated group with a pK 5.5 in the reaction mechanism, and the possible involvement of a carboxy group is corroborated by a temperature study (delta Hion = -15.9 J/mol). This, together with independent evidence from affinity-labelling experiments [Tomme, Macarrón and Claeyssens (1991) Cellulose '91, New Orleans, Abstr. 32] and n.m.r. studies [Gebbler, Gilkes, Claeyssens, Wilson, Béguin, Wakarchuk, Kilburn, Miller, Warren and Withers (1992) J. Biol. Chem. 267, 12559-12561], favours the assumption of a lysozyme-type (retention of configuration, two essential carboxy groups) mechanism for this family A cellulase.

Identification of an essential glutamate residue in the active site of endoglucanase III from Trichoderma reesei.

n-Propyl, n-butyl and n-pentyl beta-cellobiosides with a reactive omega-epoxide in their aglycon completely and irreversibly inactivate endoglucanase III from Trichoderma reesei. The pentyl derivative was found to be most effective. From these affinity labeling experiments evidence was found for the implication of Glu329 in the reaction mechanism. This is discussed in relation to other structural/functional data known for endoglucanase III and several other family A glycanases.

Mechanisms of thermoinactivation of endoglucanase I from Trichoderma reesei QM 9414.

The mechanism of irreversible thermoinactivation of endoglucanase I from Trichoderma reesei has been determined at 70 degrees C at the pH of maximum enzyme activity. The time-course of thermoinactivation did not follow first-order kinetics and kinetic constants of the process were dependent on enzyme concentration, suggesting that aggregation was the main process leading to irreversible inactivation. The enzyme was extremely resistant to urea, which in fact seemed to stabilize it against temperature. Disulphide exchange, deamidation and hydrolysis of peptide bonds were also responsible for the loss of enzyme activity at 70 degrees C.

Chemical mechanism of beta-glucosidase from Trichoderma reesei QM 9414. pH-dependence of kinetic parameters.

The variation of kinetic parameters of beta-glucosidase from Trichoderma reesei QM 9414 with pH was used to gain information about the chemical mechanism of the reaction catalysed by this enzyme. The pH-dependence of Vmax. and Vmax./Km for p-nitrophenyl beta-D-glucopyranoside showed that a group with a pK value of 4.3 must be unprotonated and a group with a pK value of 5.9 must be protonated for activity. Temperature and solvent-perturbation studies indicated that these groups are a histidine residue and a carboxy group respectively. Profiles of pKi for maltose as competitive inhibitor showed that binding is prevented when a group on the enzyme with a pK value of 4.5 becomes protonated.

Incidence of congenital rubella syndrome in 19 regions of Europe in 1980-1986.

Twenty-five cases of congenital rubella syndrome were recorded in 1,458,126 live births in 19 EUROCAT birth defects registries from 1980 to 1986. During the study period, the incidence declined steadily from 3.50 to 0.41 per 100,000 births. Rubella infection occurred in 12 multiparous women indicating failure in immunization programme.

Policies for immunization against rubella in European countries.

In 1986 a survey on immunization policies against rubella was conducted in 17 European countries. A questionnaire was sent to each Ministry of Health and selected practitioners, requesting information on official policy for vaccination against rubella, level of policy decision, immunization strategy, target groups, current recommendations for vaccination, measures taken to facilitate vaccine uptake, vaccine strains used and legislation concerning abortion. A policy for the prevention of congenital rubella is explicitly defined in 16 countries (in 12 at a national level, in 3 at a regional level and in one at both levels). In Greece only, no official policy has been defined, although the practice is to vaccinate infants over 15 months of age and girls by 9-11 years of age. There are 3 countries maintaining the selective strategy of vaccinating teenage girls, but in 8 countries this strategy changed to a mass vaccination of young infants. In 5 other countries an approach combining both mass immunization and selective vaccination is adopted. This situation of different strategies between countries in Europe could have a deleterious effect for young migrants or travelling pregnant women and could reduce the effectiveness of programmes for prevention of congenital rubella syndrome. Harmonization of immunization policies and of recommendations for vaccine administration is advisable. The mass vaccination of all young children combined with systematic revaccination either of all children or of teenage girls is probably the most effective approach for elimination of congenital rubella syndrome in Europe.