Exploring antibiotic resistant mechanism by microcalorimetry

In an effort to probe the reaction of antibiotic hydrolysis catalyzed by B3 metallo-??-lactamase (M??L), the thermodynamic parameters of penicillin G hydrolysis catalyzed by M??L L1 from Stenotrophomonas maltophilia were determined by microcalorimetric method. The values of activation free energy ??G _?? ^?? are 88.26, 89.44, 90.49, and 91.57?kJ?mol^?1 at 293.15, 298.15, 303.15, and 308.15?K, respectively, activation enthalpy ??H _?? ^?? is 24.02?kJ?mol^?1, activation entropy ??S _?? ^?? is ?219.2511?J?mol^?1?K^?1, apparent activation energy E is 26.5183?kJ?mol^?1, and the reaction order is 1.0. The thermodynamic parameters reveal that the penicillin G hydrolysis catalyzed by M??L L1 is an exothermic and spontaneous reaction.     

Exploring antibiotic resistant mechanism by microcalorimetry II

In an effort to understand the reaction of antibiotic hydrolysis with B2 metallo-??-lactamases (M??Ls), the thermodynamic parameters of imipenem hydrolysis catalyzed by metallo-??-lactamase ImiS from Aeromonas veronii bv. sobria were determined by microcalorimetric method. The values of activation free energy $ \Updelta G_{ \ne }^{\theta } $ are 86.400?±?0.043, 87.543?±?0.034, 88.772?±?0.024, and 89.845?±?0.035?kJ?mol^?1 at 293.15, 298.15, 303.15, and 308.15?K, respectively, activation enthalpy $ \Updelta H_{ \ne }^{\theta } $ is 18.586?±?0.009?kJ?mol^?1, activation entropy $ \Updelta S_{ \ne }^{\theta } $ is ?231.34?±?0.12?J?mol^?1?K^?1, apparent activation energy E is 21.084?kJ?mol^?1, and the reaction order is 1.5. The thermodynamic parameters reveal that the imipenem hydrolysis catalyzed by metallo-??-lactammase ImiS is an exothermic and spontaneous reaction.     

Enthalpies of reaction and reaction rates by flow microcalorimetry: Ester hydrolysis in basic medium

The conditions under which the Picker flow microcalorimeter can be used to measure enthalpies and rates of reactions were investigated. For this purpose, systematic studies were made of the enthalpies of neutralization of HCl, HBr, HNO ₃ , acetic, proprionic, and butyric acids with NaOH, enthalpies of hydrolysis of methyl and ethyl acetate with NaOH, and the reaction rates of the ester hydrolysis with NaOH. The general procedure and various sources of error are discussed and it is concluded that enthalpies of slow reactions can be measured to about 1% when the calorimeter is operated in the quasi-isothermal mode and the reaction rates to about 3% when operated in the quasi-adiabatic mode.On leave from Laboratoire de Thermodynamique et de Cinétique Chimique, Université de Clermont 2, Aubière 63170, France.     

Quantitation of amorphicity by microcalorimetry

The amorphous state of solids is characterized by a higher chemical and physical reactivity and a hygroscopic behaviour. Furthermore processing of amorphous powders is often difficult, because of the instability. Fast crystallizations, precipitations and milling favour the formation of the amorphous state. Galenical processes like granulation, drying, lyophilization, mixing, may also induce amorphous regions in the drug products.X-ray diffraction techniques can be used for the determination of the amorphicity of drug raw materials or drug products. Unfortunately, 10% is the detection limit, which in normal cases can be attained. Amorphous substances undergo an exothermic crystallization at temperatures above the glass transition point. Water which is a plasticizer decreases the temperature of the glass transition point, allowing the crystallization to occur at lower temperatures. The crystallization energy is measure of by microcalorimetry.Examples show the influence of the choice of the experimental conditions, especially the influence of the amorphicity on the kinetic of the reaction. Critical steps are discussed for three different drug substances. Limits of detection in the magnitude of 1 % are possible using microcalorimetry.     

Plant processing by simultaneous lactic acid fermentation and enzyme hydrolysis

Traditional ensiling of plant material by anaerobic lactic acid fermentation was combined with enzymatic hydrolysis (ENLAC for short) with cell wall degrading enzymes (hemicellulases, cellulases, and pectinases) to increase fiber digestibility or to increase the recovery of cell content from plants. Such findings were made using 0.015% (w/w, wet basis) Phylacell® enzyme preparation by ENLAC of corn and corn-sorghum mixtures, but not of forage grasses. Addition to alfalfa of a mixture of cell wall degrading enzymes, such as NOVO Viscozyme® together with NOVO Celluclast® each at 0.2–1.0% (w/w, wet basis), resulted in more rapid ensiling and improvement of rumen digestibility of silage by 20%. After 20 d of ensiling at 25 °C when the same enzymes were added to alfalfa at the 1.0% level, protein recovery by pressing increased by 35%, β-carotene recovery by 80%, and chlorophyll/xanthophyll recovery by 30%. ENLAC with the same enzymes also increased the recovery of sclareol from muscatel sage by 400%.

     

Detection of microorganisms in different growth states based on microcalorimetry

A challenging method for sterility test which was rapid and reliable had been established to assess the adaptability and robustness of the microbial under different conditions. There were material and energy metabolism or exchange with microbial on microcalorimetry, as a result this method can be served as one of the optimization of thermodynamics sterility test. Thermal power-time curves under various environmental conditions (including processing temperature, storage time, and drugs inhibition) were determined. Typical microbial growth thermal power-time curves were obtained. The curves were analyzed qualitatively and quantitatively by similarity values of bio-profiles and thermodynamics parameters, such as the exponential growth rate constant (k), detection time (T _d). The similarity showed that microbial growth curves of low processing temperature, short storage time (1?month), and Traditional Chinese Medicine injection (Shuanghuanglian, contained native compounds) inhibiting were match better with the normal than other circumstance. Thermodynamic parameters indicated that the microcalorimetric method could detect the positive bacteria within 18?h (less than 10?cfu), and more quickly identify the different states of the bacterium growth and metabolism than routine sterility. In conclusion, characterized by of the specific and strong two-dimensional information, microcalorimetry could supply thermograms as biological profiles to describe the microbial activity under different conditions, which were not only used as a rapid and reliable identification of microbial, but also as a method for sterility test of microcalorimetry optimization.     

Kinetics of enzymatic hydrolysis of lignocellulosic materials based on surface area of cellulose accessible to enzyme and enzyme adsorption on lignin and cellulose

Comparison of the model with experimental data is currently in progress. It appears that more detailed studies of the adsorption dynamics, not just adsorption equilibrium, are needed.     

Molecular mechanisms of antibiotic resistance

Over the past decade, resistance to antibiotics has emerged as a crisis of global proportion. Microbes resistant to many and even all clinically approved antibiotics are increasingly common and easily spread across continents. At the same time there are fewer new antibiotic drugs coming to market. We are reaching a point where we are no longer able to confidently treat a growing number of bacterial infections. The molecular mechanisms of drug resistance provide the essential knowledge on new drug development and clinical use. These mechanisms include enzyme catalyzed antibiotic modifications, bypass of antibiotic targets and active efflux of drugs from the cell. Understanding the chemical rationale and underpinnings of resistance is an essential component of our response to this clinical challenge.     

Thermodynamic characterization of asphaltene-resin interaction by microcalorimetry

This paper collects the work performed by isothermal titration calorimetry (ITC) to characterize the interaction between petroleum asphaltenes and resins. The interaction between these two fractions is of great interest in order to understand the mechanism of stabilization ofasphaltenes in crude oil. To simplify the approach, this preliminary study focuses on toluene solutions of both fractions. This paper reports the experimental determination of the average number of sites in asphaltene molecules and the enthalpy of interaction between asphaltenes and resins. Two models have been used to fit the experimental data. The enthalpies calculated by ITC are in the order of -2 to -4 kJ/mol. These values are in the limit of hydrogen bonding and permanent dipole energies. Similar values have been obtained by using the enthalpy as a fitting parameter in the SAFT equation.     

Study of silica-supported heteropoly acids by microcalorimetry

Silica-supported heteropoly acids have been studied by microcalorimetry. The results are greatly dependent on both the activation temperature and the heteropoly acid, in agreement with the n-hexane cracking experiments. It is shown that very strong acid sites are formed on H₄SiW₁₂O₄₀/SiO₂ activated at 623 K and on H₃PW₁₂O₄₀/SiO₂ activated at 423 K.     

The action of norfloxacin complexes on Tetrahymena investigated by microcalorimetry

Cu(nor)₂·H₂O (1), Zn(nor)₂·4H₂O (2), Ni(nor)₂·2H₂O (3), [Cu(nor)(phen)]NO₃·4H₂O (4), [Zn(nor)(phen)]NO₃·2H₂O (5), and [Ni(nor)(phen)]NO₃·3H₂O (6) were synthesized and their action on Tetrahymena growth was studied by microcalorimetry. The growth constant (k), inhibitory ratio (I), and half-inhibiting concentration (IC₅₀) were calculated, which showed that the complexes had a strong inhibitory effect on Tetrahymena. All these complexes can inhibit the growth of Tetrahymena more strongly than norfloxacin. The norfloxacin?Cmetal complexes exhibited better inhibitory activity than nor?Cphen?Cmetal complexes. The power?Ctime curves of Tetrahymena growth in the presence of norfloxacin were also measured. It was found that all complexes showed higher inhibitory activity than norfloxacin. And the inhibitory mechanism was discussed preliminarily. The diverse inhibition may be due to the ability of the complexes to penetrate into cells and the effect of these complexes on the nucleic acid. Microcalorimetry has been used extensively in many biological and chemical investigations as a universal, non-destructive, continuously running, and highly sensitive tool.     

Interaction of asphaltenes with nonylphenol by microcalorimetry

This paper shows the work performed in the study of the capability of isothermal titration calorimetry (ITC) to characterize the interaction between petroleum asphaltenes with a model molecule, namely, nonylphenol. ITC is widely used in biochemistry to study the interaction of proteins with ligands. The intention is to transfer the knowledge into the asphaltene field, with the aim of getting a better understanding of the mechanism of interaction, as well as the energies involved in this process. Calorimetric experiments show that nonylphenol has a complex mechanism of interaction with asphaltenes in toluene, including more than one process. Several models have been used to fit the experimental data. The enthalpies calculated with a model based on polymerization are in the order of -1 to -7 kJ/mol, which are very close to the hydrogen bond energies. This shows the capability of ITC to provide experimental data to the modeling of asphaltene behavior. The number of sites of interaction has been inferred by means of a model taken from protein-ligand science. The values obtained are in the range two to five sites per molecule, assuming an average Mw of 1000 units.     

Molecular structure regulation of functional polylactic acid based on chain expansion reaction and its improved hydrolysis resistance

In this work, the functional polylactic acid (PLA) was synthesized using epoxy chain extender (ADR) as a chain extender agent through melt blending method. The effects of ADR content on the molecular structure, thermal properties, and tensile properties of the functional PLA were investigated. Meanwhile, the hydrolytic behavior of the PLA/ADR materials at different hydrolysis temperatures was explored. It was found that ADR effectively regulated the molecular structure of PLA in the molten state and significantly increased the relative molecular weight, storage modulus, and complex viscosity of PLA. In addition, the Cole-Cole diagram results suggested the branched structure of PLA chain expansion systems. Based on mechanical property tests, it was noted that the addition of ADR made the molecular chain form a micro-crosslinked structure. Additionally, the mass loss rate of PLA/1.6ADR (the dosage of ADR was 1.6 wt%) was 14.75% after 14 weeks of hydrolysis under hydrolysis conditions at 58°C, while that of pure PLA was 25.89%. Moreover, the functional PLA/ADR materials exhibited significantly slower decrease rates in molecular weight, melting temperature, and tensile strength, and still maintained intact morphology after 14 weeks of hydrolysis compared to pure PLA. Therefore, the molecular structure of PLA is effectively regulated by ADR, which greatly enhances the hydrolysis resistance and further promotes the range of application of PLA.     

Enthalpies of adsorption of metal atoms on single-crystalline surfaces by microcalorimetry

The heats of adsorption of metal gas atoms onto single crystalline surfaces can be measured directly as a detailed function of coverage using a unique microcalorimeter. The development of this experimental apparatus and the results obtained from these experiments will be reviewed. The heat detection is performed with a highly sensitive pyroelectric polymer ribbon pressed into contact with the thin (1 μ m thick) single crystal sample, onto which pulses of a metal atom beam impinge. Heats of adsorption have been measured for pulses of gas containing a few per cent of a monolayer with a pulse-to-pulse standard deviation as low as 1.5 kJ · mol ^− 1. The adhesion energy of multilayer metal films can be estimated from the integral enthalpy of adsorption.     

Solid-state microcalorimetry on drug-cyclodextrin binary systems

Differential scanning calorimetry DSC has been applied to the analysis of drugcyclodextrin binary systems in order to gain experimental evidence of the interaction and determine the stoichiometry of the inclusion compound. Two model systems, paracetamolbetacyclodextrin and vinburnineg-ammacyclodextrin were tested through the comparison of thermal behaviors of interacted and non-interacted mixtures containing excess drug. DSC allowed a confirmation of both interaction and stoichiometry of the inclusion compounds.The authors wish to thank Prof. Amedeo Marini for helpful discussion and criticism.     

Study of the adsorption of n-butylamine on NaHY zeolites by microcalorimetry

The adsorption, of n-butylamine has been studied at 405 K on NaHY zeolites by microcalorimetry. The curves of the variation in the heat of adsorption as a function of the coverage change characteristically with the temperature of activation of the zeolite. From the course of these changes it is possible to establish which part of the curve corresponds to the adsorption of n-butylamine on Brönsted or Lewis acid centers.

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- 405 NaHY . . , - .

     

Hydrogen fuel electrode based on bioelectrocatalysis by the enzyme hydrogenase

Our aim is to show, that the enzymes as electrocatalysts are able to improve the performance characteristics of the fuel cells. The hydrogen fuel electrode based on hydrogenase from Thiocapsa roseopersicina immobilized directly on carbon filament material has been made. The enzyme electrode has operated according to electron tunneling between the enzyme active site and the electrode support; this mechanism is called direct bioelectrocatalysis. Under pure hydrogen the efficiencies in energy conversion of the enzyme electrode and the noble metal based commercial fuel electrode are similar concerning both the hydrogen equilibrium potential achieved and the current densities in H₂ oxidation. However, the use of the enzyme electrodes completely avoids the problem of poisoning the anode by the impurities of carbon monoxide present in reforming gas, which limits the use of cheap hydrogen containing fuel. The stability of the biological catalysts can be drastically improved by their immobilization on electrode supports, which provide the development of commercially competitive biofuel cells.