Hydrolysis Of Cephalexin Research Articles

Total turnover number prediction of an aggregating biocatalyst: Amino ester hydrolase (AEH)

Amino ester hydrolase (AEH) from Xanthomonas campestris is a promising candidate for β-lactam synthesis but suffers from low thermostability and rapid deactivation. Wild-type AEH was found to deactivate with an apparent order that varies with temperature from n = 2 at 30 °C (close to the temperature of optimum activity Topt) to n = 1.5 at 25 °C. AEH features two transitional temperatures: (1) from native ‘N’ to partially unfolded, inactive intermediate ‘I’, and (2) from partially unfolded to fully unfolded entity ‘U’ at the melting temperature Tm. CD and light scattering data suggest aggregation near Topt. To determine the total turnover number TTN, AEH was deactivated by imposing a temperature gradient and recording instantaneous rates of cephalexin hydrolysis. The TTN increased ∼ 5-fold from the wild-type (WT) for the quadruple variant N186D/A275P/E143H/V622I (QVH) at 25 °C and 10 nM AEH but varied significantly with AEH concentration and temperature.

Selectivity and kinetic modeling of penicillin G acylase variants for the synthesis of cephalexin under a broad range of substrate concentrations.

The kinetics of cephalexin synthesis and hydrolysis of the activated acyl-donor precursor phenylglycine methyl ester(PGME) were characterized under a broad range of substrate concentrations. A previously developed model by Youshko-Svedas involving the formation of the acyl-enzyme complex followed by binding of the nucleophilic β-lactam donor does not fully estimate the maximum reaction yields for cephalexin synthesis at different concentrations using initial-rate data. 7-aminodesacetoxycephalosporanic acid(7-ADCA) was discovered to be a potent inhibitor of cephalexin hydrolysis, which may account for the deviation from model predictions. Three kinetic models were compared for cephalexin synthesis, with the model incorporating competitive inhibition due to 7-ADCA yielding the best fit. Additionally, the βF24A variant and Assemblase® did not exhibit significantly different kinetics for the synthesis of cephalexin compared to the wild-type, for the concentration range evaluated and for both initial-rate experiments and time-course synthesis experiments. Lastly, a continuous stirred-tank reactor for cephalexin synthesis was simulated using the model incorporating competitive inhibition by 7-ADCA, with clear tradeoffs observed between productivity, fractional yield, and PGME conversion.

Probing the Fate of Different Structures of Beta-Lactam Antibiotics: Hydrolysis, Mineral Capture, and Influence of Organic Matter

Beta-lactam antibiotics, which are used extensively in human and veterinary applications, are commonly detected in surface waters. To examine how the distinct structures of different generations of beta-lactam antibiotics can influence their persistence or degradation in environmental aqueous media, we examined the fate of two penams (amoxicillin and cloxacillin) and two cephems (cephalexin and ceftriaxone) at pH 5.0 and pH 7.0. By contrast to the lack of hydrolysis of the penam antibiotics at both pHs, we observed hydrolysis of cephalexin at pH 7.0 (t1/2 = 12 d) and ceftriaxone at pH 5.0 (t1/2 = 2.8 d). Using high-performance liquid chromatography coupled with a diode array detector or a high-resolution mass spectrometer, we were able to confirm thiotriazinone and 3-desacetyl cefotaxime as major hydrolysis products of ceftriaxone, and propose the hydrolytic cleavage of the benzene and cephem moieties from cephalexin. In addition, we studied the effects of smectite clay particles suspended in solutions without or with dissolved organic matter. The adsorption capacity of the clay was 4- to 9-fold higher at pH 7.0 than at pH 5.0. Subsequent X-ray diffraction analysis revealed that the antibiotic adsorption was not within the clay interlayer nanopores but occurred primarily on the external clay surfaces. The addition of dissolved organic matter interfered with the adsorption of a cephem antibiotic (ceftriaxone) on the clay, but the adsorption of a penam antibiotic (amoxicillin) remained unaffected. We employed molecular modeling simulations to probe the mechanisms of adsorption on the mineral surface. Our findings offer new insights on how the compound structures can dictate different fates of the beta-lactam class of antibiotics in environmental media.

Detection of the iron complexes with hydrolysis products of cephalexin and cefradine upon high-performance liquid chromatography/electrospray ionization mass spectrometry analysis.

Cephalosporins (e.g. cephalexin, cefradine) are a major group of widely used β-lactam antibiotics. Hydrolysis of the β-lactam ring is an important reaction (often undesired) which leads to deactivation of β-lactams. To the best of our knowledge there is no electrospray ionization mass spectrometry (ESI-MS) data reported concerning the products of hydrolysis of cephalosporins. The hydrolysis of cephalexin and cefradine was performed in aqueous NaOH solutions. After the process the solutions were analyzed by high-performance liquid chromatography (HPLC)/ESI-MS. The elemental compositions of the ions discussed were confirmed by the accurate mass measurements on a quadrupole time-of-flight (QTOF) mass spectrometer. Unexpectedly, complexes between the hydrolysis products of cephalexin and cefradine (CFLh and CFRh ) and iron cation were detected upon HPLC/ESI-MS analysis, namely the ions [(CFLh -H)2 +Fe]+ and [(CFRh -H)2 +Fe]+ , although iron was not added to the analyzed solutions or to the mobile phase. These ions were found to be very stable in the gas phase. The detection of the complexes between the hydrolysis products of cephalosporins and iron may have a positive impact on the sensitivity and specificity of HPLC/ESI-MS analyses of the hydrolysis products of some cephalosporins.

Hydrolysis of cephalexin and meropenem by New Delhi metallo-β-lactamase: the substrate protonation mechanism is drug dependent

Emergence of antibiotic resistance due to New Delhi metallo-β-lactamase (NDM-1) bacterial enzymes is of great concern due to their ability to hydrolyze a wide range of antibiotics. There are ongoing efforts to obtain the atomistic details of the hydrolysis mechanism in order to develop inhibitors for NDM-1. In particular, it remains elusive how drug molecules of different families of antibiotics are hydrolyzed by NDM-1 in an efficient manner. Here we report the detailed molecular mechanism of NDM-1 catalyzed hydrolysis of cephalexin, a cephalosporin family drug, and meropenem, a carbapenem family drug. This study employs molecular dynamics (MD) simulations using hybrid quantum mechanical/molecular mechanical (QM/MM) methods at the density functional theory (DFT) level, based on which reaction pathways and the associated free energies are obtained. We find that the mechanism and the free energy barrier for the ring-opening step are the same for both the drug molecules, while the subsequent protonation step differs. In particular, we observe that the mechanism of the protonation step depends on the R2 group of the drug molecule. Our simulations show that allylic carbon protonation occurs in the case of the cephalexin drug molecule where Lys211 is the proton donor, and the proton transfer occurs via a water chain formed (only) at the ring-opened intermediate structure. Based on the free energy profiles, the overall kinetics of drug hydrolysis is discussed. Finally, we show that the proposed mechanisms and free energy profiles could explain various experimental observations.

Use of an Improved Matching Algorithm to Select Scaffolds for Enzyme Design Based on a Complex Active Site Model.

Active site preorganization helps native enzymes electrostatically stabilize the transition state better than the ground state for their primary substrates and achieve significant rate enhancement. In this report, we hypothesize that a complex active site model for active site preorganization modeling should help to create preorganized active site design and afford higher starting activities towards target reactions. Our matching algorithm ProdaMatch was improved by invoking effective pruning strategies and the native active sites for ten scaffolds in a benchmark test set were reproduced. The root-mean squared deviations between the matched transition states and those in the crystal structures were < 1.0 Å for the ten scaffolds, and the repacking calculation results showed that 91% of the hydrogen bonds within the active sites are recovered, indicating that the active sites can be preorganized based on the predicted positions of transition states. The application of the complex active site model for de novo enzyme design was evaluated by scaffold selection using a classic catalytic triad motif for the hydrolysis of p-nitrophenyl acetate. Eighty scaffolds were identified from a scaffold library with 1,491 proteins and four scaffolds were native esterase. Furthermore, enzyme design for complicated substrates was investigated for the hydrolysis of cephalexin using scaffold selection based on two different catalytic motifs. Only three scaffolds were identified from the scaffold library by virtue of the classic catalytic triad-based motif. In contrast, 40 scaffolds were identified using a more flexible, but still preorganized catalytic motif, where one scaffold corresponded to the α-amino acid ester hydrolase that catalyzes the hydrolysis and synthesis of cephalexin. Thus, the complex active site modeling approach for de novo enzyme design with the aid of the improved ProdaMatch program is a promising approach for the creation of active sites with high catalytic efficiencies towards target reactions.

Preparation of Poly (N-Cephalexin Amic Acids) as Drug Polymers

In this work new monomers have been prepared such as N-Cephalexin maleamic acid M1 and N-Cephalexin citriconamic acid M2 , from reaction of Cephalexine with maleic anhydride and citraconic anhydride at room temperature using dioxane as a solvent.The new prepared monomers M1and M2 were polymerized free radically with AIBN as initiator to their corresponding poly amic acids P1 and P2. Whichwere converted to their sodium salt polymers P3 and P4 to enhance their solubility in water.The physical and chemical properties were studied; the prepared monomers and polymers were characterized by FTIR, 1H-NMR and UV. Spectroscopy, theintrinsic viscosity was measured by Ostwald viscometer at 30 0C with DMF as a solvent; the drug release rate was studied. Experimental results showed that the hydrolysis of Cephalexin in alkaline medium was higher than acidic medium.&#x0D; The softening point of the prepared P1 drug polymer was 130.23-138.350C with-190.17mJ as used in softing point,and softening point of the prepared P2 was 207-2200C

Cobalt(II)-promoted hydrolysis of cephalexin: Crystal structure of the cephalosporoate–cobalt(II) complex

The cobalt(II) ion-promoted hydrolysis of the widely used cephalosporin antibiotic cephalexin is confirmed by solid crystallographic characterization of the cephalosporoate–cobalt(II) complex [Co(cephalosporoate) 2] · 6H 2O ( 1), obtained from an aqueous solution containing Co(OAc) 2 and cephalexin. The crystal structure analysis results prove that the double bond of dihydrothiazine ring in cephalosporoate undergoes a shift from position 3–4 to 4–5 in aqueous media at low pH, with the consequent uptake of a proton at C(3) by the α face.

Modeling of the enzymatic kinetic synthesis of cephalexin--influence of substrate concentration and temperature.

During enzymatic kinetic synthesis of cephalexin, an activated phenylglycine derivative (phenylglycine amide or phenylglycine methyl ester) is coupled to the nucleus 7-aminodeacetoxycephalosporanic acid (7-ADCA). Simultaneously, hydrolysis of phenylglycine amide and hydrolysis of cephalexin take place. This results in a temporary high-product concentration that is subsequently consumed by the enzyme. To optimize productivity, it is necessary to develop models that predict the course of the reaction. Such models are known from literature but these are only applicable for a limited range of experimental conditions. In this article a model is presented that is valid for a wide range of substrate concentrations (0-490 mM for phenylglycine amide and 0-300 mM for 7-ADCA) and temperatures (273-298 K). The model was built in a systematic way with parameters that were, for an important part, calculated from independent experiments. With the constants used in the model not only the synthesis reaction but also phenylglycine amide hydrolysis and cephalexin hydrolysis could be described accurately. In contrast to the models described in literature, only a limited number (five) of constants was required to describe the reaction at a certain temperature. For the temperature dependency of the constants, the Arrhenius equation was applied, with the constants at 293 K as references. Again, independent experiments were used, which resulted in a model with high statistic reliability for the entire temperature range. Low temperatures were found beneficial for the process because more cephalexin and less phenylglycine is formed. The model was used to optimize the reaction conditions using criteria such as the yield on 7-ADCA or on activated phenylglycine. Depending on the weight of the criteria, either a high initial phenylglycine amide concentration (yield on 7-ADCA) or a high initial 7-ADCA concentration (yield on phenylglycine amide) is beneficial.

Immobilization of penicillin G acylase onto chemically grafted nylon particles

Nylon particles, grafted with diethylene glycol dimethacrylate (DGDA) using potassium persulphate as initiator, were treated with hexamethylene diamine (HMDA). The aminoalkylated particles were activated with glutaraldehyde and finally, penicillin G acylase (PA) was immobilized to these activated particles. Both the conditions of the aminoalkylation- and the immobilization process were optimized. The hydrolysis of cephalexin was used as model conversion. The retention of activity of the immobilized enzyme was 12%. This value improved to 30% by adding phenyl acetic acid (PAA), as active-site protecting agent, to the enzyme solution. The results suggest formation of multi-point attachment between the enzyme and the matrix.

Non-isothermal cephalexin hydrolysis by penicillin G acylase immobilized on grafted nylon membranes

A new catalytic membrane has been prepared using a nylon membrane grafted by γ-radiation with methylmethacrylate (MMA) and using hexamethylenediamine (HMDA) as spacer. Penicillin G acylase (PGA) and cephalexin were employed as catalyst and substrate, respectively. Cephalexin hydrolysis was studied in bioreactors operated under isothermal and non-isothermal conditions. A hydrolysis increase was found when the temperature of the warm membrane surface was kept constant and the temperature of the other membrane surface was kept at a lower value. The hydrolysis increase was linearly proportional to the applied temperature difference. Cephalexin hydrolysis increased to about 10% when a temperature difference of 1°C was applied across the catalytic membrane. These results have been attributed to the non-isothermal cephalexin transport across the membrane, i.e., to the process of thermodialysis. In this way, the enzyme immobilized on and into the membrane reacts with a substrate concentration higher than that produced by simple diffusion under isothermal conditions.

Characterization of the activity of penicillin G acylase immobilized onto nylon membranes grafted with different acrylic monomers by means of γ-radiation

Penicillin G acylase (PGA) has been immobilized onto nylon membranes grafted with methylmethacrylate (MMA) or diethyleneglycoldimethacrylate (DGDA) monomers by means of γ-radiation. Hexamethylenediamine (HMDA) has been used as spacer between the grafted membranes and the enzyme. Glutaraldehyde (GA) was used as crosslinking to couple the enzyme to the HMDA. The catalytic membranes so prepared were studied as a function of pH and temperature of the solution containing the substrate. The membranes showing the best characteristics were the ones grafted with DGDA. The dependence of the behavior of these membranes on several experimental conditions was studied, i.e., the temperature and duration of the aminoalkylation process, spacer concentration, the glutaraldehyde concentration and the enzyme concentration. The experimental conditions giving the best performance of the catalytic membranes have been deduced. The time requested to obtain 50% of substrate conversion, i.e., hydrolysis of cephalexin, has been studied as a function of its initial concentration.

The Use of Chromogenic Reference Substrates for the Kinetic Analysis of Penicillin Acylases

Determination of kinetic parameters of penicillin acylases for phenylacetylated compounds is complicated due to the low Km values for these substrates, the lack of a spectroscopic signal, and the strong product inhibition by phenylacetic acid. To overcome these difficulties, a spectrophotometric method was developed, with which kinetic parameters could be determined by measuring the effects on the hydrolysis of the chromogenic reference substrate 2-nitro-5-[(phenylacetyl)amino]benzoic acid (NIPAB). To that end, spectrophotometric progress curves with NIPAB in the absence and presence of the phenylacetylated substrates and their products were measured and analyzed by numerical fitting to the appropriate equations for competing substrates with product inhibition. This analysis yielded kinetic constants for phenylacetylated substrates such as penicillin G, which are in close agreement with those obtained in independent initial velocity experiments. Using NIPAB analogs with lower kcat/Km values, kinetic parameters for the hydrolysis of cephalexin and penicillin V were determined. This method was suitable for determining the kinetic constants of penicillin acylases in periplasmic extracts from Escherichia coli, Alcaligenes faecalis, and Kluyvera citrophila. The use of chromogenic reference substrates thus appears to be a rapid and reliable method for determining kinetic constants with various substrates and enzymes.

Use of aqueous two-phase systems for in situ extraction of water soluble antibiotics during their synthesis by enzymes immobilized on porous supports

Yields of kinetically controlled synthesis of antibiotics catalyzed by penicillin G acylase from Escherichia coli (PGA) have been greatly increased by continuous extraction of water soluble products (cephalexin) away from the surroundings of the enzyme. In this way its very rapid enzymatic hydrolysis has been avoided. Enzymes covalently immobilized inside porous supports acting in aqueous two-phase systems have been used to achieve such improvements of synthetic yields. Before the reaction is started, the porous structure of the biocatalyst can be washed and filled with one selected phase. In this way, when the pre-equilibrated biocatalyst is mixed with the second phase (where the reaction product will be extracted), the immobilized enzyme remains in the first selected phase in spite of its possibly different natural trend. Partition coefficients (K) of cephalexin in very different aqueous two-phase systems were firstly evaluated. High K values were obtained under drastic conditions. The best K value for cephalexin (23) was found in 100% PEG 600-3 M ammonium sulfate where cephalexin was extracted to the PEG phase. Pre-incubation of immobilized PGA derivatives in ammonium sulfate and further suspension with 100% PEG 600 allowed us to obtain a 90% synthetic yield of cephalexin from 150 mM phenylglycine methyl ester and 100 mM 7-amino desacetoxicephalosporanic acid (7-ADCA). In this reaction system, the immobilized enzyme remains in the ammonium sulfate phase and hydrolysis of the antibiotic becomes suppressed because of its continuous extraction to the PEG phase. On the contrary, synthetic yields of a similar process carried out in monophasic systems were much lower (55%) because of a rapid enzymatic hydrolysis of cephalexin.

β-Lactamase-catalysed hydrolysis of cephalexin: evolution of the cephalosporoate intermediate

Hydrolysis of cephalexin catalysed by P99 β-lactamase from Enterobacter cloacae has been studied at pD 6.4 and 8.0. The enzymatic hydrolysis product is the cephalosporoate intermediate, which in aqueous media undergoes tautomerization of the double bond in the dihydrothiazine ring from position 3–4 to 4–5, with the consequent uptake of a proton at C(3). This latter process is governed by steric constraints. NMR results show that the cephalosporoate compound bears the aromatic group, present in the side chain at C(7), folded over the dihydrothiazine ring. Under these conditions the proton is chiefly taken up by the α face of the dihydrothiazine ring. Subsequently, the six-membered ring can be subjected to several reactions according to the pH.

Reaction kinetics of cephalexin synthesizing enzyme from Xanthomonas citri

In enzymatic synthesis of cephalexin from D-alpha-phenylglycine methyl ester (PGM) and 7-amino-3-deacetoxy-cephalosporanic acid (7-ADCA) using alpha-acylamino-beta-lactam acylhydrolase from Xanthomonas citri, it was found that this enzyme catalyzes all three reactions including PGM hydrolysis, cephalexin synthesis, and cephalexin hydrolysis. Based on our experimental results, a mechanistic kinetic model for cephalexin synthesizing enzyme system having acyl-enzyme intermediate was proposed. From this kinetic model, the reaction rate equations for three reactions were derived, and the kinetic parameters were evaluated. A good agreement between the simulation results and the experimental results was found.

Cephalexin synthesis by partially purified and immobilized enzymes

AbstractAn enzyme which catalyzes the synthesis of cephalexin fromD‐α phenylglycinemethylester (PGM) and 7‐amino‐3‐desacetoxy‐cephalosporanic acid (7‐ADCA) was prepared from Xanthomonas citri (IFO 3835) and partially purified 30‐fold by ammonium sulfate fractionation, DEAE‐cellulose, and Sepharose‐4B column chromatography. The Km values for 7‐ADCA, PGM, and cephalexin were determined as 11.1, 2.1, and 1.61 mM, respectively. The enzymatic cephalexin synthesis follows the reversible bi‐uni reaction kinetics. The equilibrium constant is influenced by the initial mole ratios of 7‐ADCA and PGM. The cephalexin hydrolysis is catalyzed by the same cephalexin synthesizing enzyme, but methanol does not participate in the hydrolytic reaction. The amount of enzyme in the reaction mixture affects the initial rate but does not influence the equilibrium product concentration. This cephalexin‐synthesizing enzyme was immobilized onto several adsorbents. Among these, Kaolin and bentonite showed a higher retention of enzyme activity and stability for reuse. The immobilized‐enzyme reaction kinetics were investigated and compared with those of the soluble enzyme. A rate expression for the enzymatic synthesis of cephalexin was derived. The results of computer simulation showed good agreement with the experimental results.

ChemInform Abstract: HYDROLYSIS OF CEPHALOSPORINS IN STRONGLY ACID MEDIUM

The hydrolysis of cephalexin, cephalothin and cephaloridine in strongly acid medium (10--25% sulfuric acid) at high temperature (approximately 100 degrees C) was studied. The hydrolysis proceeds with a high rate (10--15 min are necessary for complete hydrolysis) and results in definite products with low mol.-wt. The hydrolysis under such hard conditions affects the side chain in 3-position, in some cases the C--N bond in the amido group in 7-position, and leads to a total cleavage of the beta-lactam ring, giving thiazine compounds. The reaction products were identified using IR and UV spectral data, thin-layer and gas chromatography, and some characteristic reactions of functional groups. The results of the investigation were used for development of photometric procedures for determination of cephalosporins.

Fluorometric determination of cephalexin in urine.

An intensively fluorescent product was formed in alkaline solution by hydrolysis of cephalexin at elevated temperature. The fluorescent product gave excitation and emission maxima at 355 and 435 nm, respectively. It was readily extracted into ethyl acetate and a acetone-chloroform mixture from an acidic solution and then extracted back into an alkaline solution. Using this new method, urinary excretion behaviors in human volunteers after oral administration of cephalexin capsule were investigated.

Enzymatic synthesis of cphalosporins. III. Purification and properties of penicillin acylase from Kluyvera citrophila.

Penicillin acylase (EC 3. 5. 1. 11) of Kluyvera citrophila KY7844 was purified approximately 120-fold by DEAE-cellulose chromatography, hydroxyapatite chromatography and isoelectrofocusing fractionation. The purified enzyme, with an approximate molecular weight of 63, 000, appeared to be homogeneous in disc electrophoretic analysis, and showed isoelectric point (Ip) 8.12 and 13.0 units/mg of specific activity for cephalexin hydrolysis. The Michaelis constant (Km) for cephalexin and for 7-[1-(1H)-tetrazolylacetamido]-desacetoxycephalosporanic acid ((1H) T-7ADCA) was 1.4mm and 3.6mm, respectively. This enzyme was capable of producing (1H) T-7ADCA in 80% yield from 1-(1H)-tetrazolylacetate methylester and 7-aminodesaceto-xycephalosporanic acid.