Joëlle Saulnier

Elastolytic activity of Pseudomonas aeruginosa elastase

Elastolysis of insoluble elastin by Pseudomonas aeruginosa elastase was found to be less specific (higher apparent Km value) but more active (higher activity) than with pancreatic elastase. Furthermore, pancreatic and P. aeruginosa elastases act synergistically during the initial stages of elastolysis. After extensive hydrolysis, the size distribution of digestion products was lower with P. aeruginosa than with pancreatic elastase. The higher extent of hydrolysis may be explained by the fact that, if pancreatic elastase needs at least six sub-sites for activity, P. aeruginosa elastase may hydrolyse tetrapeptides such as tetraalanine, or synthetic substrates such as furylacryloyltripeptides FA-X-Leu-Y, X and Y being Gly and/or Ala.

Capillary electrophoresis in the assay of the hydrolysis of glycine-containing peptides by a protease from Pseudomonas aeruginosa

A rapid and simple capillary zone electrophoresis (CZE) method for measuring the activity of a minor protease from Pseudomonas aeruginosa is described. When glycine-containing oligopeptides were used as substrates, it was possible to separate and quantify substrate and products. Moreover oligopeptide hydrolysates were analysed by fast atom bombardment mass spectrometry providing the sites of splitting of the substrates. By comparison with CZE calibration curves constructed with reference peptides, the initial rates of hydrolysis were calculated. The method, validated for pentaglycine hydrolysis, was also used for the analysis of reaction mixtures and for monitoring the enzymic hydrolysis of various peptides in order to investigate enzyme specificity.

ON THE MECHANISM OF ACTION OF PSEUDOLYSIN: KINETIC STUDY OF THE ENZYMATIC CONDENSATION OF Z-Ala WITH Phe-NH2

We previously demonstrated that pseudolysin, a neutral metalloproteinase from thermolysin family, is a suitable catalyst for peptide bond formation through reverse proteolysis. In order to get an insight into the yield- and rate-controlling factors, we studied the kinetics and mechanism of pseudolysin-catalysed synthesis. The condensation of Z-Ala-Phe-NH2 from Z-Ala and Phe-NH2, performed in a semi-organic homogeneous system, was chosen as a reaction model. We applied the conductimetric method for measuring the initial rates of the enzymatic reaction before product precipitation. We have both optimised synthesis and conductimetric conditions. Highest velocities were obtained in the pH range 7.0-7.5. When kept below 0.15, ionic strength did not significantly alter the rate values. Kinetic studies were then performed for varied Z-Ala and Phe-NH2 concentrations at different, fixed concentrations of the alternative substrate, at pH 7.3 in 35% MeOH and for an ionic strength below 0.15. Graphical inspection and numerical analysis of initial-rate data permitted calculation of the kinetic parameters and proposition of a kinetic mechanism for pseudolysin. The values of kinetic constants were: 6.5 mM Z-Ala-Phe-NH2 synthesised/min/μM pseudolysin (Vm), 70 mM (KM of Z-Ala) and 180mM (KM of Phe-NH2). In addition, the condensation reaction proceeded via a rapid-equilibrium random bireactant mechanism, as was previously demonstrated for thermolysin, another extensively studied metalloprotease.

Conductimetric assay of pyroglutamyl peptidase activity

Abstract A conductimetric method was applied to assay Streptococcus pyogenes pyroglutamyl peptidase (E.C. 3.4.8.11) activity using pyroglutamylalanine or pyroglutamyltyrosine. A spectrophotometric comparison using pyroglutamyl-β-naphthylamide indicated that the sensitivity was much increased when using conductimetry. The use of a C-protected dipeptide such as pyroglutamylalanineamide also increased the sensitivity. Kinetic parameters have been measured for both dipeptides, with KM values of 0.34 mM and 0.47 mM, respectively, for the alanine and tyrosine derivatives.

Comparison of carboxypeptidase Y and thermolysin for ochratoxin A electrochemical biosensing

Herein, carboxypeptidase Y (CPY) and thermolysin (TLN) were compared as sensing elements to develop an original biosensor for the direct detection of ochratoxin A (OTA) in olive oil. Electrochemical detection of OTA was performed using biosensors containing either CPY or TLN immobilized through glutaraldehyde vapor cross-linking onto gold interdigitated microelectrodes. Different parameters affecting biosensor sensitivity towards OTA were first optimized. TLN and CPY exhibited an optimal activity under the same conditions: 35 min cross-linking time, working pH of 7 and temperature of 25 °C. TLN and CPY biosensors exhibited comparable analytical performances. The conductometric response of both modified electrodes was linear up to 75 μM. The limits of detection were also very similar (1 μM for the CPY biosensor and 0.7 μM for the TLN biosensor). For both enzymes, initial rate vs. OTA concentration plots could be described by hyperbolic curves consistent with the Michaelis–Menten model. KappM and Vappmax, deduced from these curves, were 26 μM and 3.3 μS min−1 respectively for the TLN biosensor. The biosensors signal was reproducible, relative standard deviations calculated from three consecutive measurements of standards in the 1–75 μM range, being lower than 5%. The signals were also very stable over a 30 days period when biosensors were stored at 4 °C in 20 mM phosphate buffer between two measurements. The TLN biosensor was further evaluated for OTA determination in spiked olive oil samples. Recovery values were close to 100% demonstrating the suitability of this method for OTA screening in olive oil. Interestingly, OTA concentrations could be determined without pretreatment of the sample and no matrix effect was observed.

Nanomaterial-based electrochemical biosensors for food safety and quality assessment

One of the key challenges to both control foodstuff composition and detect chemical or biological contaminants in food is the availability of selective, sensitive, and reliable analytical methods. In this context, electrochemical biosensors are particularly attractive due to their low-cost, high sensitivity, and selectivity. They can be easily miniaturized and integrated in high density arrays for multianalyte and high throughput analysis. To improve their sensitivity and robustness, different strategies have been developed, including the incorporation of nanomaterials such as gold or magnetic nanoparticles, carbon-based nanostructures (eg, nanotubes, graphene). These nano-objects offer high surface areas and can be used as platforms to enhance bioreceptors loading and stability, also serving as relays for electron transfer between the biomolecules and the electrode. In this chapter, new trends in nanomaterial-based electrochemical biosensors for food analysis are reviewed, focusing on the determination of major contaminants, that is, foodborne pathogens, toxins, veterinary drug residues, and pesticides.

Age-dependent variations of human PMNL elastase activity as a function of pH, ionic strength and calcium concentration

Human leukocyte elastase (HLE) has been implicated in the pathomechanism of various diseases, such as emphysema and atherosclerosis. The incidence of these diseases is increasing with aging. Therefore, it can be supposed that the HLE activity is changing with aging according to the well known age-related physiological alterations. Thus, the effects of pH, NaCl and calcium concentrations on HLE activity, separated from polymorphonculear leucocytes (PMNLs) of healthy, young (<35 years) and elderly (>75 years) subjects, were studied by measuring the activity with synthetic substrate and with bovine and human atherosclerotic and non-atherosclerotic aortic elastin. From our results, it may be concluded, that the elastolytic activity of HLE separated from PMNLs of elderly subjects is more sensitive to ionic strength, to pH and to the calcium concentration of the medium, than the HLE activity of young subjects. The elastolytic activity of HLE, of both young and old subjects, increases dramatically on atherosclerotic aortic elastin in the presence of calcium. These findings might explain, at least partly, the increased incidence of atherosclerosis with aging.

Use of conductimetry for the monitoring of thermodynamically-controlled enzymatic peptide synthesis

A conductimetric method was developed for the monitoring of thermodynamically-controlled enzyme peptide synthesis. It was applied to the synthesis of Nα-benzyloxycarbonylalanylphenylalaninamide, in the presence of Pseudomonas aeruginosa elastase. A linear continuous decrease of conductance could be demonstrated in the first minutes of the reaction, allowing a precise measurement of the initial rate of synthesis. Using independent calibration graphs, the conductimetric method correlated well with liquid chromatography, the most often applied technique for enzymatic peptide synthesis monitoring. Conductimetry may therefore be considered as a promising technique for quantitative measurements of such reactions.

Responsive Polydiacetylene Vesicles for Biosensing Microorganisms

Polydiacetylene (PDA) inserted in films or in vesicles has received increasing attention due to its property to undergo a blue-to-red colorimetric transition along with a change from non-fluorescent to fluorescent upon application of various stimuli. In this review paper, the principle for the detection of various microorganisms (bacteria, directly detected or detected through the emitted toxins or through their DNA, and viruses) and of antibacterial and antiviral peptides based on these responsive PDA vesicles are detailed. The analytical performances obtained, when vesicles are in suspension or immobilized, are given and compared to those of the responsive vesicles mainly based on the vesicle encapsulation method. Many future challenges are then discussed.