Synthesis Of Cephalexin Research Articles

Mathematical analysis of batch reactor performance for the enzymatic synthesis of cephalexin: Laplace Homotopy perturbation method and Adomian decomposition method

In this article, a mathematical model of diffusion reaction in kinetically controlled cephalexin synthesis in the batch reactor with penicillin acylase immobilized in glyoxyl-agarose is analyzed. The kinetic model is a non-linear non-stead-state reaction–diffusion equation with non-linear terms related to the Fick’s law. We have presented the approximate analytical expression for the non-steady-state non-linear reaction–diffusion equation by utilizing the Laplace homotopy perturbation method (LHPM) and for steady-state by using LHPM and Adomian decomposition method (ADM). The approximate analytical solution obtained from these methods proved that they are fit for every values of the reaction–diffusion and kinetic parameters. We also present the numerical solution of the considered reaction–diffusion equation by using pdepe tool in MATLAB software. When comparing the semi-analytical solution with the numerical solution, a satisfactory result is noted for all the possible values of the parameters. The closed-form analytical expressions for the effectiveness factor in non-steady and steady-state conditions are obtained and analyzed using LHPM and ADM.

Improvement of α-amino Ester Hydrolase Stability via Computational Protein Design.

Amino ester hydrolases (AEHs) are capable of rapid synthesis of cephalexin but suffer from rapid deactivation even at low temperatures. Previous efforts to engineer AEH have generated several improved variants but have been limited in scope in part due to limitations in activity assay throughput for β-lactam synthesis reactions. Rational design of 'whole variants' was explored to rapidly improve AEH thermostability by mutating between 3-15% of residues. Most variants were found to be inactive due to a mutated calcium binding site, the function of which has not previously been described. Four active variants, all with improved melting temperatures, were characterized in terms of synthesis and hydrolysis activity, melting temperature, and deactivation at 25°C. Two variants were found to have improved total turnover numbers relative to the initial AEH variant; however, a clear tradeoff exists between improved stability and overall activity of each variant.

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.

Reactor Design and Optimization of α-Amino Ester Hydrolase- Catalyzed Synthesis of Cephalexin.

Pharmaceutical production quality has recently been a focus for improvement through incorporation of end-to-end continuous processing. Enzymatic ß-lactam antibiotic synthesis has been one focus for continuous manufacturing, and α-amino ester hydrolases (AEHs) are currently being explored for use in the synthesis of cephalexin due to their high reactivity and selectivity. In this study, several reactors were simulated to determine how reactor type and configuration impacts reactant conversion, fractional yield toward cephalexin, and volumetric productivity for AEH-catalyzed cephalexin synthesis. The primary reactor configurations studied are single reactors including a continuous stirred-tank reactor (CSTR) and plug flow reactor (PFR) as well as two CSTRS and a CSTR + PFR in series. Substrate concentrations fed to the reactors as well as enzyme concentration in the reactor were varied. The presence of substrate inhibition was found to have a negative impact on all reactor configurations studied. No reactor configuration simultaneously allowed high substrate conversion, high fractional yield, and high productivity; however, a single PFR was found to enable the highest substrate conversion with higher fractional yields than all other reactor configurations, by minimizing substrate inhibition. Finally, to further demonstrate the impact of substrate inhibition, an AEH engineered to improve substrate inhibition was simulated and Pareto optimal fronts for a CSTR catalyzed with the current AEH were compared to Pareto fronts for the improved AEH. Overall, reduced substrate inhibition would allow for high substrate conversion, fractional yield, and productivity with only a single CSTR.

Synthesis, Characterization and Biological Evaluation Study of Cephalexin (Ceph) and Isatin Schiff base and Its Complexation with Some Divalent Metal Ions

Synthesis, Characterization and Biological Evaluation Study of Cephalexin (Ceph) and Isatin Schiff base and Its Complexation with Some Divalent Metal Ions

Enhanced Dissolution of 7-ADCA in the Presence of PGME for Enzymatic Synthesis of Cephalexin.

Enzymatic catalysis has been recognized as a green alternative to classical chemical route for synthesis of cephalexin (CEX). However, its industrial practice has been severely limited by the low productivity due to the low solubility of 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and high hydrolysis of D-phenylglycine methyl ester (PGME). In this work, the enhanced dissolution of 7-ADCA in the presence of PGME for efficient enzymatic synthesis of CEX was investigated. Results showed that the solubility of 7-ADCA in water could be improved by PGME. Moreover, supersaturated solution of 7-ADCA could be created in the presence of PGME by a pH shift strategy. The supersaturated solution of 7-ADCA possess good stability, which could be explained in terms of the inhibition of 7-ADCA precipitation due to the presence of PGME. The interaction between 7-ADCA and PGME is explored by spectroscopic determination and DFT analysis and the mechanism of enhanced dissolution of 7-ADCA in the presence of PGME is discussed and proposed. The feasibility of supersaturated solution of 7-ADCA for the enzymatic synthesis of CEX is evaluated. It was demonstrated that high conversion ratio (> 95.0%) and productivity (> 240.0mmol/L/h) was obtained under a wide range of reaction conditions, indicating that the supersaturated solution system was highly superior to conventional homogeneous solution system. The information obtained in this work will be helpful to industrial production of CEX via enzymatic route.

Kinetic model development for α-amino ester hydrolase (AEH)-catalyzed synthesis of β-lactam antibiotics

α-Amino ester hydrolases (AEH) are a class of enzymes highly specific for catalyzing the synthesis and hydrolysis of amides and esters of α-amino acids. They are particularly promising catalyst candidates as an alternative to penicillin G acylase (PGA) for synthesis of a variety of β-lactam antibiotics, including cephalexin, cefadroxil, amoxicillin, and ampicillin. In this study, X. campestris pv. campestris AEH and two previously established quadruple variants designed for enhanced stability (N186D/A275P/V622I/E143G termed QVG and N186D/A275P/V622I/E143H termed QVH) were characterized in terms of their kinetic properties. AEH appears to have highest substrate specificity for cephalosporins, particularly cephalexin. The optimum pH value for activity was found to be between pH 6.5 – 6.75 for synthesis of amoxicillin, cephalexin, and cefadroxil, while synthesis selectivity consistently increased with decreasing pH value. Evidence for substrate inhibition preventing the β-lactam nucleus from binding and forming the corresponding amide is discussed. Time-course kinetic data across a wide range of substrate concentrations was found to corroborate the presence of substrate inhibition, and five possible kinetic models are derived to explain AEH-catalyzed cephalexin synthesis kinetic profiles. A best fit model that incorporates both competitive substrate inhibition and partial substrate inhibition was selected based on the Akaike Information Criterion (AIC) and quality of fit at high substrate concentration. Finally, progress of the synthesis of beta-lactams in a single CSTR was simulated using the best fit kinetic model and a Pareto optimal front was constructed for fractional yield, productivity, and substrate conversion to evaluate potential reactor configurations.

Enzyme synthesis of cephalexin in continuous-flow microfluidic device in ATPS environment

We present an experimental study dealing with the transfer of the enzyme synthesis of cephalexin from a batch arrangement to a continuous-flow microfluidic system with integrated reaction product separation and enzyme recovery. Cephalexin is synthesized by penicillin acylase in a kinetic regime that is characterized by the appearance of a concentration maximum during the enzyme reaction. We determine proper reaction conditions providing a high cephalexin yield and relatively short reaction time by performing a set of batch experiments. Once the residence/reaction time was found, the reaction process is transferred into a continuous mode. We employ an aqueous two-phase system (ATPS) forming two-phase slug flow in a microfluidic capillary as the reaction-separation environment. Such a flow arrangement guarantees a uniform residence time of the reaction mixture in the reaction microcapillary, and it provides in situ extraction of cephalexin. ATPS also offers an easy way for enzyme recycling and addition of fresh reactants as a benefit. We optimized the composition of the ATPS based on phosphates, polyethylene glycol and water so that cephalexin showed a high affinity to one phase and a soluble enzyme to the other phase. Due to the low level of utilization of a soluble catalyst in the continuous flow arrangement, we recycled the reaction phase containing the dissolved enzyme. The recycling loop also contained microdialysis unit for removal of phenylglycine that tends to cause system clogging. We show that the final design of the microfluidic system with recycle can operate continuously for at least 5 h. Our integrated microfluidic platform represents a general solution for enzymatic reactions performed with simultaneous reaction product separation and enzyme recycle.

Efficient enzymatic synthesis of cephalexin in suspension aqueous solution system.

An efficient method for the enzymatic synthesis of cephalexin (CEX) from 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and d-phenylglycine methyl ester (PGME) using immobilized penicillin G acylase (IPGA) as catalyst in a suspension aqueous solution system was developed, where the reactant 7-ADCA and product CEX are mainly present as solid particles. The effects of key factors on the enzymatic synthesis were investigated. Results showed that continuous feeding of PGME was more efficient for the synthesis of CEX than the batch mode. Under the optimized conditions, the maximum 7-ADCA conversion ratio of 99.3% and productivity of 200 mmol/L/H were achieved, both of which are much superior to the homogeneous aqueous solution system. Besides, IPGA still retained 95.4% of its initial activity after 10 cycles of enzymatic synthesis, indicating the excellent stability of this approach. The developed approach shows great potential for the industrial production of CEX via an enzyme-based route.

Theoretical study on enzyme synthesis of cephalexin in a parallel-flow microreactor combined with electrically driven ATPS microextraction

A mathematical model of a microfluidic device with two aqueous phases for the simultaneous cephalexin production and its separation from a reaction mixture was developed. The model anticipates the continuous cephalexin synthesis and enzyme recyclation.

Crystallization Kinetics of Cephalexin Monohydrate in the Presence of Cephalexin Precursors

The solubility and crystallization kinetics of cephalexin monohydrate, an important β-lactam antibiotic, are reported for the first time for an aqueous solution. The nucleation and growth kinetics are determined with online process analytical technologies (PAT) and offline image analysis to support implementation of a continuous process. Effects of two cosolutes, 7-aminodesacetoxycephalosporanic acid (7-ADCA) and phenylglycine methyl ester (PGME), on nucleation and growth are examined. These cosolutes are precursors in the enzymatic synthesis of cephalexin. Primary nucleation of cephalexin was unaffected by the cosolutes, but 7-ADCA, even in small quantities (<10 mM), significantly decreased the cephalexin monohydrate growth rate; PGME had a less significant impact on growth rate. Possible mechanisms of growth inhibition are discussed; a model wherein cosolutes slow growth by pinning advancing growth steps and halting step advancement below a critical supersaturation best explains the data. Impact on crys...

Development of a Polyaniline-coated Monolith Reactor for the Synthesis of Cephalexin Using Penicillin G Acylase Aggregates

A monolith reactor for the synthesis of cephalexin was developed using capillary columns. The micro channel in the monolith reactor was coated with polyaniline (PANI), and penicillin G acylase was aggregated with PANI using 0.5% of glutaraldehyde as a cross-linker. The developed monolith reactor exhibited many advantages over other enzyme reactors such as batch and continuous reactors. It showed fast enzyme reaction rates owing to the decrease in external mass transfer and internal diffusion limitations. The reactor can easily be scaled up by bundling together multiple monolith reactors, enabling a corresponding increase in feed rate. Furthermore, the monolith reactor showed good operational stability, with 95% of its original activity maintained after 48 h of continuous operation. The PANI coating on the surface of the capillary column increased the enzyme immobilization capacity and conversion was increased from 15.4% to 70.6% after PANI coating. The conversion ratio increased to approximately 70.6% with an increase in residence time and reactor length.

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.

Design of a sensitive fluorescent polarization immunoassay for rapid screening of milk for cephalexin.

In this paper we describe the development of a sensitive, fast, and easily performed fluorescence polarization immunoassay for determination of cephalexin in milk. The experimental work was performed to increase sensitivity and specificity. Therefore, the structures of the tracers were varied by synthesis of both cephalexin (CEX) and cephalotin (CET) conjugates with a variety of fluorescent labels. Two rabbit antisera containing antibodies against cephalexin and cephalotin were tested in homologous and heterologous combinations with the tracers. For every working antibody-tracer combination, the analytical conditions and cross-reactivity for structural analogues-cephalosporins and other antibiotics that could also be present in milk-were determined. It was found that the highest sensitivity was achieved by use of the homologous pair CET-EDF-anti-CET antibody (limit of detection (LOD) 0.4 μg kg(-1) for standard solutions prepared in buffer), but this combination was not appropriate because of high cross-reactivity with CET. For subsequent experiments, therefore, CEX- EDF-anti-CEX antibody were chosen (LOD 0.8 μg kg(-1) for standard solutions prepared in buffer). Part of this manuscript is devoted to the variation of precipitation agents for pretreatment of milk before analysis; milk is an extremely complicated matrix. The optimum protein precipitation agent was methanol. This technique for cephalexin determination was characterized by a limit of detection of 1 μg kg(-1). The method was validated by using naturally contaminated and spiked milk samples. The results obtained corresponded very well with those obtained by HPLC, which was used as confirmation method.

Synthesis and application of cephalexin imprinted polymer for solid phase extraction in milk

Molecular imprinted polymer (MIP) against cephalexin was synthesized by co-polymerization of functional monomer, cross-linker, radical initiator, along with target molecule (cephalexin) in a porogenic material. Binding of cephalexin towards prepared MIP was studied in different solvents (water, methanol, 1M NaCl, acetone and acetonitrile) and best binding was observed in methanol. Partition coefficient and selectivity of prepared imprint and non-imprint was also studied. Cross reactivity in terms of binding efficiency was also assessed with other antibiotics. Chromatographic study of MIP was carried out by packing prepared imprint into glass column. MIP was used as matrix in solid phase extraction (SPE) for recovery of cephalexin from spiked milk samples for further estimation by high performance liquid chromatography. No interference was observed from milk components after elution of cephalexin from MIP, indicating selectivity and affinity of MIP. On the other hand, interference was observed in eluate obtained from C18 SPE column.

Enzymatic synthesis of Cephalexin in recyclable aqueous two-phase systems composed by two pH responsive polymers

The synthesis of Cephalexin in novel recyclable aqueous two-phase systems was studied, using immobilized penicillin acylase as biocatalyst, 7-amino-3-desacetoxy cephalosporanic acid (7-ADCA) as nucleophile and phenyl glycine methyl ester (PGME) as acyl donor. The aqueous two-phase system (ATPS) was composed by two pH-response copolymers PADBA and PMDB, which could be recycled efficiently. In the ATPS, high partition coefficient (2.57) for Cephalexin and low partition coefficient (0.21) for 7-ADCA were obtained. Then the synthesis of Cephalexin in ATPS and conventional one-phase system was compared. Higher Cephalexin yield was obtained in ATPS. In addition, synthesis in ATPS was improved at higher substrates concentrations, obtaining yield of 98.2% with 50mM 7-ADCA and 150mM PGME at pH 5.8, 20°C. Even reducing acyl donor to nucleophile ratio to 2, the yield can still above 90%. PADBA/PMDB recycling aqueous two-phase system is suitable for synthesis of Cephalexin.

Recombinant alpha-amino ester acid hydrolase from Xanthomonas rubrilineans VKPM B-9915 is a highly efficient biocatalyst of cephalexin synthesis

Recombinant, as well as native alpha-amino acid ester hydrolase from Xanthomonas rubrilineans VKPM B-9915 (XrAEH, EC 3.1.1.43), was tested for synthesis of amino-beta-lactam antibiotic cephalexin. It was shown that the recombinant enzyme r-XrAEH produced by Escherichia coli VKPM B-11246 is more efficient in comparison with the native enzyme wt-XrAEH prepared from mutant strain Xanthomonas rubrilineans VKPM B-9915. When r-XrAEH was used as a biocatalyst, addition of ethylene glycol (33 vol %) to the reaction medium improved the yield from 70 to 95%. During synthesis of cephalexin under optimal conditions in the case of the native enzyme wt-XrAEH the cephalexin yield was 85%, in contrast to r-XrAEH where it was 95%. Furthermore, unlike native wt-XrAEH enzymes, preparations of recombinant r-XrAEH do not possess beta-lactamase side activity.

Comparative study of the enzymatic synthesis of cephalexin at high substrate concentration in aqueous and organic media using statistical model

Synthesis of cephalexin with immobilized penicillin acylase at high substrates concentration at an acyl donor to nucleophile molar ratio of 3 was comparatively evaluated in aqueous and ethylene glycol media using a statistical model. Variables under study were temperature, pH and enzyme to substrate ratio and their effects were evaluated on cephalexin yield, ratio of initial rates of cephalexin synthesis to phenylglycine methyl ester hydrolysis, volumetric and specific productivity of cephalexin synthesis, that were used as response parameters. Results obtained in both reaction media were modeled using surface of response methodology and optimal operation conditions were determined in terms of an objective function based on the above parameters. At very high substrates concentrations the use of organic co-solvents was not required to attain high yields and actually almost stoichiometric yields were obtained in a fully aqueous media with the advantages of higher productivities than in an organic co-solvent media and compliance with the principles of green chemistry.

Efficient biocatalyst for large-scale synthesis of cephalosporins, obtained by combining immobilization and site-directed mutagenesis of penicillin acylase

We describe the rational design of a new efficient biocatalyst and the development of a sustainable green process for the synthesis of cephalosporins bearing a NH₂ group on the acyl side chain. The new biocatalyst was developed starting from the WT penicillin acylase (PA) from Escherichia coli by combining enzyme mutagenesis, in position α146 and β24 (βF24A/αF146Y), and immobilization on an appropriate modified industrial support, glyoxyl Eupergit C250L. The obtained derivative was used in the kinetically controlled synthesis of cephalexin, cefprozil and cefaclor and compared to the WT-PA and an already described mutant, PA-βF24A, with improved properties. The new biocatalyst posses a very high ratio between the rates of the synthesis and two undesired hydrolyses (acylating ester and the amidic product). In particular, a very low amidase activity was observed with PA-βF24A/αF146Y and, consequently, the hydrolysis of the produced antibiotic was avoided during the process. Taking advantage of this property, higher conversions in the synthesis of cephalexin (99% versus 76%), cefaclor (99% versus 65%) and cefprozil (99% versus 60%) were obtained compared to the WT enzyme. Furthermore, the new mutant also show a higher synthetic activity compared to PA-βF24A immobilized on the same support, allowing the maximum yields to be achieved in very short reaction times. The production of cephalexin with the immobilized βF24A/αF146Y acylase has been developed on a pre-industrial scale (30 l). After 20 cycles, the average yield was 93%. The biocatalyst showed good stability properties and no significant decrease in performance.

Batch reactor performance for the enzymatic synthesis of cephalexin: influence of catalyst enzyme loading and particle size

A mathematical model is presented for the kinetically controlled synthesis of cephalexin that describes the heterogeneous reaction-diffusion process involved in a batch reactor with glyoxyl-agarose immobilized penicillin acylase. The model is based on equations considering reaction and diffusion components. Reaction kinetics was considered according to the mechanism proposed by Schroën, while diffusion of the reacting species was described according to Fick's law. Intrinsic kinetic and diffusion parameters were experimentally determined in independent experiments. It was found that from the four kinetic constants, the one corresponding to the acyl-enzyme complex hydrolysis step had the greatest value, as previously reported by other authors. The effective diffusion coefficients of all substances were about 5×10(-10)m(2)/s, being 10% lower than free diffusion coefficients and therefore agreed with the highly porous structure of glyoxyl-agarose particles. Simulations made from the reaction-diffusion model equations were used to evaluate and analyze the impact of internal diffusional restrictions in function of catalyst enzyme loading and particle size. Increasing internal diffusional restrictions decreases the Cex synthesis/hydrolysis ratio, the conversion yield and the specific productivity. A nonlinear relationship between catalyst enzyme loading and specific productivity of Cex was obtained with the implication that an increase in catalyst enzyme loading will not increase the volumetric productivity by the same magnitude as it occurs with the free enzyme. Optimization of catalyst and reactor design should be done considering catalyst enzyme loading and particle size as the most important variables. The approach presented can be extended to other processes catalyzed by immobilized enzymes.