Synthesis Of Ampicillin Research Articles

Synthesis of new Ampicillin supported on Cr/Oxidized pectin supported on Ti bimetal–organic framework as an efficient antimicrobial and anticancer nanocomposite

To synthesize, develop and provide advanced materials with unique properties, in this study, new bimetal–organic framework nanocomposites using Titanium, chromium, oxidized petin and ampicillin were synthesized. With TGA/DTG, FT-IR, XRD, XPS, BET, and EDAX mapping, the structure of the newly synthesized compound was identified, and its structural features were reported. The thermal stability up to 200 °C, particle size of 82 nm and the specific surface area of 27.700 m2/g was from the specifications of the final product. Antimicrobial properties of different bacterial and fungal species and anticancer properties on breast cancer cells were evaluated. In biological evaluation, the synthesized nanocomposites have unique activity and was more effective than some commercial drugs. In antimicrobial activities, MIC was observed between 2–64 μg/mL and in anticancer properties IC50 of 183 μg/mL.

Efficient synthesis of β-lactam antibiotics with in situ product removal by a newly isolated penicillin G acylase.

A penicillin G acylase (PGA) from Achromobacter xylosoxidans PX02 was newly isolated, and site-directed mutagenesis at three important positions αR141, αF142, βF24 was carried out for improving the enzymatic synthesis of β-lactam antibiotics. The efficient mutant βF24A was selected, and the (Ps/Ph)ini (ratio between the initial rate of synthesis and hydrolysis of the activated acyl donor) dramatically increased from 1.42-1.50 to 23.8-24.1 by means of the optimization of reaction conditions. Interestingly, the efficient enzymatic synthesis of ampicillin (99.1% conversion) and amoxicillin (98.7% conversion) from a high concentration (600mM) of substrate 6-APA in the low acyl donor/nucleus ratio (1.1:1) resulted in a large amount of products precipitation from aqueous reaction solution. Meanwhile, the by-product D-phenylglycine was hardly precipitated, and 93.5% yield of precipitated ampicillin (561mM) and 94.6% yield of precipitated amoxicillin (568mM) were achieved with high purity (99%), which significantly simplified the downstream purification. This was the first study to achieve efficient β-lactam antibiotics synthesis process with in situ product removal, with barely any by-product formation. The effect enzymatic synthesis of antibiotics in aqueous reaction solution with in situ product removal provides a promising model for the industrial semi-synthesis of β-lactam antibiotics.

Enzymatic reactive crystallization for improving ampicillin synthesis

The enzyme penicillin G acylase (PGA) catalyzes the condensation of phenylglycine methyl ester (PGME) with 6-aminopenicillanic acid (6-APA) to form ampicillin. We improved the selectivity of ampicillin synthesis with PGA by running simultaneous reaction and crystallization. However, the enzyme also catalyzes two undesirable side reactions: the hydrolysis of PGME to phenylglycine and the hydrolysis of ampicillin to phenylglycine and 6-APA. We demonstrate that a fifty percent improvement in selectivity for ampicillin over phenylglycine is achieved by combining reaction and crystallization in batch at pH value of 6 with saturated 6-APA and equimolar PGME. The enhancement in selectivity is mainly attributed to the decreased rates of enzymatic ampicillin hydrolysis; however, the course of the pH value during the reaction also has an effect on enzyme activity that improved selectivity. In addition to showing experimental results, we developed a new kinetic process model that predicts the observed improvement. The new model accounts for the solubility limits of different species as functions of pH value as well as the large change in pH value at high conversion. Previous work does not account for changes in activity with conversion. The pH-dependent activity for the specific enzyme used in this system, Assemblase® from DSM-Sinochem, is well-realized by the model and generalization to other PGAs is possible within the model framework; the selectivity parameters α, β0, and γ for Assemblase® are compared to PGA from E. coli as evidence.

Design, synthesis and microbiological evaluation of ampicillin-tetramic acid hybrid antibiotics.

Exploiting iron-uptake pathways by conjugating β-lactam antibiotics with iron-chelators such as catechol and hydroxamic acid is a proven strategy to overcome permeability-related resistance in Gram-negative bacteria. Since naturally occurring iron chelating tetramic acids have not been previously examined for this purpose, an exploratory series of novel ampicillin-tetramic acid hybrids that structurally resemble ureidopenicillins was designed and synthesized. The new analogs were evaluated for the ability to chelate iron and their MIC activities determined against a representative panel of clinically significant bacterial pathogens. The tetramic acid β-lactam hybrids demonstrated a high affinity to iron in the order of 10−30 M3. The hybrids were less active against Gram-positive bacteria. However, against Gram-negative bacteria, their activity was species dependent with several hybrids displaying improved activity over ampicillin against wild-type Pseudomonas aeruginosa. The anti-Gram-negative activities of the hybrids improved in the presence of clavulanic acid revealing that the tetramic acid moiety did not provide added protection against β-lactamases. Additionally, the hybrids were found to be efflux pump substrates as their activities markedly improved against pump-inactivated strains. Unlike the catechol and hydroxamic acid siderophore β-lactam conjugates, the activities of the hybrids did not improve under iron-deficient conditions. These results suggest that the tetramic acid hybrids gain permeability via different membrane receptors, or they are out competed by native bacterial siderophores with stronger affinities for iron. This study provides a foundation for the further exploitation of the tetramic acid moiety to achieve novel β-lactam anti-Gram-negative agents, providing that efflux and β-lactamase mediated resistance is addressed.

Efficient enzymatic synthesis of ampicillin using mutant Penicillin G acylase with bio-based solvent glycerol

To fulfill the industry demand of ampicillin enzymatic synthesis, immobilized mutant Penicillin G acylase and bio-based solvent glycerol were employed at high substrate concentration and low acyl donor/nucleophile ratio. After process optimization, good yield and low operation costs were achieved.

Efficient cascade synthesis of ampicillin from penicillin G potassium salt using wild and mutant penicillin G acylase from Alcaligenes faecalis

To avoid isolation and purification of the intermediate 6-aminopenicillanic acid (6-APA), a two-enzyme two-step cascade synthesis of ampicillin from penicillin G was established. In purely aqueous medium, penicillin G hydrolysis and ampicillin synthesis were catalyzed by immobilized wild-type and mutagenized penicillin G acylases from Alcaligenes faecalis (Af PGA), respectively (Fig. 1). The βF24 G mutant Af PGA (the 24th Phenylalanine of the β-subunit was replaced by Glycine) was employed for its superior performance in enzymatic synthesis of ampicillin. By optimizing the reaction conditions, including enzyme loading, temperature, initial pH and d-PGME/6-APA ratio, the conversion of the second step of ampicillin synthesis reached approximately 90% in 240min and less than 1.7mole d-PGME were required to produce 1mole ampicillin. Overall, in a 285min continuous two-step procedure, an ampicillin yield of 87% was achieved, demonstrating the possibility of improving the cascade synthesis of ampicillin by mutagenized PGA, providing an economically efficient and environmentally benign procedure for semi-synthetic penicillins antibiotics synthesis.

Efficient enzymatic synthesis of ampicillin by mutant Alcaligenes faecalis penicillin G acylase

Semi-synthetic β-lactam antibiotics (SSBAs) are one of the most important antibiotic families in the world market. Their enzymatic synthesis can be catalyzed by penicillin G acylases (PGAs). In this study, to improve enzymatic synthesis of ampicillin, site-saturating mutagenesis was performed on three conserved amino acid residues: βF24, αR146, and αF147 of thermo-stable penicillin G acylase from Alcaligenes faecalis (Af PGA). Four mutants βF24G, βF24A, βF24S, and βF24P were recovered by screening the mutant bank. Kinetic analysis of them showed up to 800-fold increased kcat/Km value for activated acyl donor D-phenylglycine methyl ester (D-PGME). When βF24G was used for ampicillin synthesis under kinetic control at industrially relevant conditions, 95% of nucleophile 6-aminopenicillanic acid (6-APA) was converted to ampicillin in aqueous medium at room temperature while 12% process time is needed to reach maximum product accumulation at 25% enzyme concentration compared with the wild-type Af PGA. Consequently, process productivity of enzymatic synthesis of ampicillin catalyzed by Af PGA was improved by more than 130 times, which indicated an enzyme viable for efficient SSBAs synthesis.

In situ mixed donor synthesis of ampicillin with ethylene glycol co‐solvent

Ethylene glycol has the ability to act as a reactant when employed as a co-solvent for the enzymatic synthesis of ampicillin from (R)-phenylglycine methyl ester and 6-aminopenicillanic acid. The side reaction positively affects yield because its product, (R)-phenylglycine hydroxyethyl ester, is an intermediate for ampicillin synthesis.

Enzymatic Synthesis of Ampicillin by Immobilized Penicillin G acylase of E.coli

Enzymatic Synthesis of Ampicillin by Immobilized Penicillin G acylase of E.coli

Improving the diastereoselectivity of penicillin G acylase for ampicillin synthesis from racemic substrates

Semi-synthetic β-lactam antibiotics are synthesized enzymatically with the use of penicillin G acylase (PGA). Currently, PGA only exhibits weak diastereoselectivity with respect to the alpha amino group of rac-phenylglycine methyl ester (rac-PGME) when it is coupled with 6-aminopenicillanic acid to synthesize ampicillin. Therefore, we sought to improve the diastereoselectivity of PGA by targeting residues for site-saturation based on the proximity to the substrate's chiral center. Four variants with improved selectivity for (R)-ampicillin synthesis were identified, all resulting from a mutation at the β24 position. βPhe24Ala, while not identified from our library screening, was obtained with site-directed mutagenesis because it has been previously shown to be selective for (R)-enantiomers with substituents other than an amino group. The diastereomeric excess (d.e.(R)) value of 37% for the wild-type enzyme was improved to a d.e.(R) value of 98% for our most selective mutant, βPhe24Ala. Also, four mutations at the α146 position that resulted in (S)-selective PGA variants were identified. βPhe24 and αPhe146 are on opposite sides of the alpha carbon of the substrate, and we have shown that altering these residues results in enhanced selectivity in opposite directions. All variants that showed selectivity for (S)-ampicillin synthesis showed decreased synthetic activity for pure substrates and a decreased synthesis-to-hydrolysis ratio. In contrast, the mutants that were selective for (R)-ampicillin showed significantly decreased primary and secondary hydrolysis when synthesizing ampicillin from pure (R)-PGME, resulting in up to 4-fold decrease in the synthesis to hydrolysis ratio and up to 2-fold increase in the yield achieved. Finally, it was discovered that the selective PGA variants have racemase or epimerase activity, a fascinating phenomenon that has never been reported.

Enzymatic Synthesis of Ampicillin: Nonlinear Modeling, Kinetics Estimation, and Adaptive Control

Nowadays, the use of advanced control strategies in biotechnology is quite low. A main reason is the lack of quality of the data, and the fact that more sophisticated control strategies must be based on a model of the dynamics of bioprocesses. The nonlinearity of the bioprocesses and the absence of cheap and reliable instrumentation require an enhanced modeling effort and identification strategies for the kinetics. The present work approaches modeling and control strategies for the enzymatic synthesis of ampicillin that is carried out inside a fed-batch bioreactor. First, a nonlinear dynamical model of this bioprocess is obtained by using a novel modeling procedure for biotechnology: the bond graph methodology. Second, a high gain observer is designed for the estimation of the imprecisely known kinetics of the synthesis process. Third, by combining an exact linearizing control law with the on-line estimation kinetics algorithm, a nonlinear adaptive control law is designed. The case study discussed shows that a nonlinear feedback control strategy applied to the ampicillin synthesis bioprocess can cope with disturbances, noisy measurements, and parametric uncertainties. Numerical simulations performed with MATLAB environment are included in order to test the behavior and the performances of the proposed estimation and control strategies.

Ampicillin Synthesis Using a Two-Enzyme Cascade with Both α-Amino Ester Hydrolase and Penicillin G Acylase.

The current enzymatic production of semisynthetic β-lactam antibiotics requires isolation and purification of the intermediate 6-aminopenicillanic acid which adds cost and complexity to the manufacturing process. In this work, we took advantage of the unique substrate specificity of a-amino ester hydrolases to perform a purely aqueous one-pot production of ampicillin from penicillin G and D-phenylglycine methyl ester, catalyzed by α-amino ester hydrolase and penicillin G acylase. The synthesis was performed in both a one-pot, one-step synthesis resulting in a maximum conversion of 39%, and a one-pot, two-step process resulting in a maximum conversion of 47%. The two-enzyme cascade reported in this paper is a promising alternative to the current enzymatic two-step, two-pot manufacturing process for semisynthetic β-lactam antibiotics which requires intermittent isolation of 6-aminopenicillanic acid.

An efficient synthesis of ampicillin on magnetically separable immobilized penicillin G acylase

Penicillin G acylase (PGA) was immobilized on the magnetic hydrophilic polymer microspheres with average pore size of 17.1 nm, specific surface area of 128.2 m 2/g and saturate magnetization of 6.4 emu/g. The 96.7% ampicillin yield with 1.60 of the synthesis/hydrolysis (S/H) ratio from 6-aminopenicillanic acid (6-APA) and d-(−)-alpha-phenylglycine methyl ester ( d-PGME) can be achieved using the resultant magnetic biocatalyst in ethylene glycol, where only 82.1% yield with 1.40 of the S/H ratio was obtained using the free PGA under the identical reaction conditions. The immobilized PGA can be separated magnetically and recycled for five times without obvious loss of its catalytic activity.

Solid–liquid equilibrium of substrates and products of the enzymatic synthesis of ampicillin

AbstractThe solid–liquid equilibrium of precursors and products of the enzymatic synthesis of ampicillin (AMP) [6‐aminopencillanic acid (6‐APA) and D(−)phenylglycine (PG)] was investigated at different temperatures (283–298 K) and pHs (5.5–7.5). Solubility data were obtained using an analytical methodology. Equilibrium dissociation constants were experimentally measured at several temperatures for AMP, 6‐APA, PG, and D(−)phenylglycine methyl ester. A model based on the simplified perturbed hard sphere theory proposed by Khoshkbarchi and Vera (Ind Eng Chem Res. 1996;35:4319‐4327) was fitted against solubility data. The model could describe the water solubility behavior for AMP and PG as function of pH and temperature, but a bias was observed when fitting the model to the solubility of 6‐APA. © 2009 American Institute of Chemical Engineers AIChE J, 2010

Enzymatic synthesis of ampicillin in precipitated medium with different acyl donor derivatives: perspectives for industrial application

Enzymatic synthesis of ampicillin in precipitated medium with different acyl donor derivatives: perspectives for industrial application

A two-step, one-pot enzymatic synthesis of ampicillin from penicillin G potassium salt

A two-step, one-pot synthesis of ampicillin from penicillin G potassium salt (PGK) in aqueous buffer/organic co-solvent has been achieved. Ethylene glycol (EG) was chosen as the organic co-solvent. Factors including co-solvent content, enzyme loading, reaction temperature and substrate concentration were investigated. The optimum conditions were as follow: pH 8.0 phosphate buffer solution, 50% EG (v/v), 25 °C, 100 mM PGK and 300 mM d-phenylglycine methyl ester (D-PGM), 43.2 IU/ml IPA-750. The maximum yield was 57.3% after a reaction time of 17 h. It is the first report about the synthesis of ampicillin from penicillin G potassium salt in one-pot combining the enzymatic hydrolysis and the subsequent enzymatic condensation, and the novel methodology will have important application in the β-lactam antibiotics industry.

Cloning of penicillin acylase from Escherichia coli: Catalytic properties of recombinant enzymes

The gene of penicillin acylase (PA) from Escherichia coli has been cloned from a PA producer strain that is an analogue of strain ATCC 11105. Optimization of the cultivation conditions made it possible to obtain up to 130 mg of active enzyme per liter of culture broth. A number of single, double, and triple mutants were obtained by the method of site-specific mutagenesis using PCR. As a result of isolation and purification procedures, homogeneous preparations of the wild-type enzyme and its mutants were obtained. Studies showed that (1) the obtained enzymes have the correctly folded structure; (2) complexing agents and metal cations do not inhibit their catalytic activity; (3) mutant PAs, like the wild type, are efficiently inactivated by phenylmethylsulfonyl fluoride (PMSF), which makes it possible to titrate their active sites; and (4) the obtained mutants are characterized by a greater specificity constant in the reaction of hydrolysis of a colorimetric substrate; however, they are inferior to the wild type in the synthesis of ampicillin by acyl transfer.

Efficient enzymatic synthesis of ampicillin in organic media

The kinetically controlled synthesis of ampicillin with immobilized penicillin acylase (IPA) from Escherichia coli in fully organic medium was studied. d-phenylglycine methyl ester ( d-PGM) was selected as the activated acyl donor due to its good solubility in organic solvents. A series of organic solvents with different polarity were screened and ethyl acetate was found to be the most satisfying solvent for the enzymatic synthesis of ampicillin. Remarkable catalytic activity of the IPA was retained in ethyl acetate, and high yield could be obtained. Furthermore, some significant factors that greatly affect the ampicillin synthesis process, such as substrate concentration, temperature and water content of IPA etc., were investigated in details. As a result, high yield (92.9%) and synthesis/hydrolysis (S/H) ratio (1.50) were successfully achieved under the optimum conditions.

Influence of mass transfer limitations on the enzymatic synthesis of β-lactam antibiotics catalyzed by penicillin G acylase immobilized on glioxil-agarose

Mass transfer effects were investigated for the synthesis of ampicillin and amoxicillin, at pH 6.5 and 25 degrees C, catalyzed by penicillin G acylase immobilized on agarose. The influence of external mass transfer was analysed using different stirring rates, ranging form 200 to 800 rpm. Above 400 rpm, the film resistance may be neglected. Intra-particle diffusion limitation was investigated using biocatalysts prepared with different enzyme loads and agarose with different mean pore diameters. When agarose with 6, 8 and 10% of crosslinking were used, for the same enzyme load, substrates and products concentration profiles presented no expressive differences, suggesting pore diameter is not important parameter. An increase on enzyme load showed that when more than 90 IU of enzyme activity were used per mL of support, the system was influenced by intra-particle mass transfer. A reactive-diffusive model was used to estimate effective diffusivities of substrates and products.

Nonconventional Reactor for Enzymatic Synthesis of Semi-Synthetic β-Lactam Antibiotics

The enzymatic synthesis of β-lactam semi-synthetic antibiotics has been receiving increasing attention as a green-chemistry alternative for the industrial production of these drugs, because mild reaction conditions may be used. A nonconventional fed-batch reactor is presented here, using a bi-disperse gel matrix for immobilization of the enzyme penicillin G acylase (PGA) [EC 3.5.1.11]. The catalyst particles are suspended within Taylor−Couette vortices, performing the kinetically controlled synthesis of ampicillin (AMP) from phenylglycine methyl ester (PGME) and 6-aminopenicillanic acid (6-APA). This is a serial−parallel set of reactions, where the desired product (AMP) is the intermediate species, and a high selectivity is essential for the process economics. With this objective, AMP should be precipitated, withdrawing the antibiotic from the liquid phase and reducing its hydrolysis. One key point is to protect the physical integrity of the catalyst within this environment. To avoid damages to the cataly...