Synthesis Of Amoxicillin Research Articles

Investigation of Enzyme Immobilization and Clogging Mechanisms in the Enzymatic Synthesis of Amoxicillin.

This study investigated the blocking mechanism of immobilized penicillin G acylase (PGA) during the enzymatic synthesis of amoxicillin. Laboratory observations revealed that the primary cause of clogging was the crystallization of the substrate and product on the enzyme surface. Adjusting key parameters can significantly reduce clogging and improve catalytic efficiency. Methanol can decrease enzyme activity, but isopropyl alcohol cleaners can effectively remove clogs and protect enzyme activity. These findings provide an experimental foundation for optimizing the PGA immobilization process, which is crucial for achieving high efficiency and sustainability in industrial production.

Parvicella tangerina gen. nov., sp. nov. (Parvicellaceae fam. nov., Flavobacteriales), first cultured representative of the marine clade UBA10066, and Lysobacter luteus sp. nov., from activated sludge of a seawater-processing wastewater treatment plant.

Two strains isolated from a sample of activated sludge that was obtained from a seawater-based wastewater treatment plant on the southeastern Mediterranean coast of Spain have been characterized to achieve their taxonomic classification, since preliminary data suggested they could represent novel taxa. Given the uniqueness of this habitat, as this sort of plants are rare in the world and this one used seawater to process an influent containing intermediate products from amoxicillin synthesis, we also explored their ecology and the annotations of their genomic sequences. Analysis of their 16S rRNA gene sequences revealed that one of them, which was orange-pigmented, was distantly related to Vicingus serpentipes (family Vicingaceae) and to other representatives of neighbouring families in the order Flavobacteriales (class Flavobacteriia) by 88-89 % similarities; while the other strain, which was yellow-pigmented, was a putative new species of Lysobacter (family Xanthomonadaceae, order Xanthomonadales, class Gammaproteobacteria) with Lysobacter arseniciresistens as closest relative (97.3 % 16S rRNA sequence similarity to its type strain). Following a polyphasic taxonomic approach, including a genome-based phylogenetic analysis and a thorough phenotypic characterization, we propose the following novel taxa: Parvicella tangerina gen. nov., sp. nov. (whose type strain is AS29M-1T=CECT 30217T=LMG 32344T), Parvicellaceae fam. nov. (whose type genus is Parvicella), and Lysobacter luteus sp. nov. (whose type strain is AS29MT=CECT 30171T=LMG 32343T).

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.

Poly-lysine supported cross-linked enzyme aggregates of penicillin G acylase and its application in synthesis of β-lactam antibiotics

Penicillin G acylase (PGA) from Providencia rettgeri PX04 (PrPGA) was utilized to synthesize β-lactam antibiotics. Poly-lysine supported cross-linked enzyme aggregates (PL-CLEAs) were prepared using PGA. Addition of poly-lysine significantly increased retention of PGA activity in CLEAs, with a decrease in the synthesis/hydrolysis (S/H) ratio. PL-CLEAs with 0.56 mg/mL poly-lysine retained 83% of free PGA activity, and displayed a higher S/H ratio than that of the free enzyme. Both PL-CLEAs and CLEAs exhibited high pH and thermal stabilities. PL-CLEAs possessed the best stability profile, and the lowest α value [(kcat/Km)Ps/(kcat/Km)AD], and was most effective at amoxicillin synthesis. A >94% yield of amoxicillin was achieved using a D-HPGME/6-APA ratio of 1.2:1 (240 mM, 200 mM), with fed-batch addition of D-HPGME. PL-CLEAs displayed excellent operational stability during amoxicillin synthesis. Over 97% of initial conversion was retained after twenty rounds of catalysis. PL-CLEAs exhibited greater potency than CLEAs in practical catalysis, permitting a higher concentration of reactants.

Dynamic Modelling and Optimisation of the Batch Enzymatic Synthesis of Amoxicillin

Amoxicillin belongs to the β-lactam family of antibiotics, a class of highly consumed pharmaceutical products used for the treatment of respiratory and urinary tract infections, and is listed as a World Health Organisation (WHO) “Essential Medicine”. The demonstrated batch enzymatic synthesis of amoxicillin is composed of a desired synthesis and two undesired hydrolysis reactions of the main substrate (6-aminopenicillanic acid (6-APA)) and amoxicillin. Dynamic simulation and optimisation can be used to establish optimal control policies to attain target product specification objectives for bioprocesses. This work performed dynamic modelling, simulation and optimisation of the batch enzymatic synthesis of amoxicillin. First, kinetic parameter regression at different operating temperatures was performed, followed by Arrhenius parameter estimation to allow for non-isothermal modelling of the reaction network. Dynamic simulations were implemented to understand the behaviour of the design space, followed by the formulation and solution of a dynamic non-isothermal optimisation problem subject to various product specification constraints. Optimal reactor temperature (control) and species concentration (state) trajectories are presented for batch enzymatic amoxicillin synthesis.

Efficient synthesis of β-lactam antibiotics with very low product hydrolysis by a mutant Providencia rettgeri penicillin G acylase.

Penicillin G acylase (PGA) was isolated from Providencia rettgeri PX04 (PrPGApx04) and utilized for the kinetically controlled synthesis of β-lactam antibiotics. Site-directed mutagenesis was performed to increase the process efficiency. Molecular docking was carried out to speculate the key mutant positions corresponding with synthetic activity, which resulted in the achievement of an efficient mutant, βF24G. It yielded higher conversions than the wild-type enzyme in the synthesis of amoxicillin (95 versus 17.2%) and cefadroxil (95.4 versus 43.2%). The reaction time for achieving the maximum conversion decreased from 14 to 16h to 2-2.5h. Furthermore, the secondary hydrolysis of produced antibiotics was hardly observed. Kinetic analysis showed that the (kcat/Km)AD value for the activated acyl donor D-hydroxyphenylglycine methyl ester (D-HPGME) increased up to 41 times. In contrast, the (kcat/Km)Ps values for the products amoxicillin and cefadroxil decreased 6.5 and 21 times, respectively. Consequently, the α value (kcat/Km)Ps/(kcat/Km)AD, which reflected the relative hydrolytic specificity of PGA for produced antibiotics with respect to the activated acyl donor, were only 0.028 and 0.043, respectively. The extremely low hydrolytic activity for the products of the βF24G mutant enabled greater product accumulation to occur during synthesis, which made it a promising enzyme for industrial applications.

Solubility measurements of amoxicillin in mixtures of water and ethanol from 283.15 to 298.15 K

Among the various types of existing antibiotics, amoxicillin stands out as a semi synthetic penicillin widely used on a world scale. The enzymatic synthesis of amoxicillin has arisen as an alternative to the drawbacks of conventional chemical synthesis. Nevertheless, to make it feasible a wide data basis of solubility is required in different conditions of industrial interest, aiming at crystallization and subsequent purification of the antibiotic. In this case, the variables such as temperature, pH and solvent composition are of fundamental importance. This work presents solubility data of amoxicillin in aqueous media using an analytical method. The solubility measurements were carried out at 283.15–298.15 K, varying the pH between 2 and 8, and ethanol composition up to 70 wt%. Dissociation constants have also been measured at the studied values of temperature and ethanol composition, using potentiometric titration. The values of pKa demonstrated to be in agreement with the solubility behavior observed experimentally. Thus, the ideal model based on pKa values has been applied as reference for the representation of the solubility data. Furthermore, the experimental solubility and pKa data sets can be used for correlation and thermodynamic modeling purposes.

Covalent Immobilization of Penicillin G Acylase onto Fe3O4@Chitosan Magnetic Nanoparticles.

Penicillin G acylase (PGA) was immobilized on magnetic Fe3O4@chitosan nanoparticles through the Schiff base reaction. The immobilization conditions were optimized as follows: enzyme/support 8.8 mg/g, pH 6.0, time 40 min, and temperature 25°C. Under these conditions, a high immobilization efficiency of 75% and a protein loading of 6.2 mg/g-support were obtained. Broader working pH and higher thermostability were achieved by the immobilization. In addition, the immobilized PGA retained 75% initial activity after ten cycles. Kinetic parameters Vmax and Km of the free and immobilized PGAs were determined as 0.91 mmol/min and 0.53 mmol/min, and 0.68 mM and 1.19 mM, respectively. Synthesis of amoxicillin with the immobilized PGA was carried out in 40% ethylene glycol at 25°C and a conversion of 72% was obtained. These results showed that the immobilization of PGA onto magnetic chitosan nanoparticles is an efficient and simple way for preparation of stable PGA.

In situ one-pot preparation of superparamagnetic hydrophilic porous microspheres for covalently immobilizing penicillin G acylase to synthesize amoxicillin

Magnetic hydrophilic porous microspheres were successfully one-pot synthesized for the first time via in situ inverse suspension polymerization of glycidyl methacrylate, N,N′-methylene bisacrylamide and 2-hydroxyethyl methacrylate in the presence of Fe3+ and Fe2+ dispersed in formamide, which were denoted as magnetic Fe3O4-GMH microspheres. The morphology and properties of magnetic Fe3O4-GMH microspheres were characterized by SEM, VSM, XRD, FTIR, and so on. The formamide content had an important influence on the morphology of Fe3O4-GMH, and nearly perfectly spherical Fe3O4-GMH particles were formed when the amount of formamide was 15ml. The diameters of the microspheres were in the range of 100–200μm and Fe3O4-GMH exhibited superparamagnetic behavior with the saturation magnetization of 5.44emu/g. The specific surface area of microspheres was 138.7m2/g, the average pore diameter and pore volume were 15.1nm and 0.60cm3/g, respectively. The content of oxirane groups on Fe3O4-GMH was 0.40mmol/g. After penicillin G acylase (PGA) was covalently immobilized on Fe3O4-GMH microspheres, the catalytic performance for amoxicillin synthesis by 6-aminopenicillanic acid and d-hydroxyphenylglycine methyl ester was largely improved. As a result, 90.1% amoxicillin yield and 1.18 of the synthesis/hydrolysis (S/H) ratio were achieved on PGA/Fe3O4-GMH with ethylene glycol as solvent, but only 62.6% amoxicillin yield and 0.37 of the S/H ratio were obtained on free PGA under the same reaction conditions. Furthermore, the amoxicillin yield and S/H ratio were still kept at 88.2% and 1.06, respectively after the immobilized PGA was magnetically separated and recycled for 10 times, indicating that PGA/Fe3O4-GMH had a very good reusability.

Penicillin G acylase from Achromobacter sp. CCM 4824

Penicillin G acylase from Achromobacter sp. (NPGA) was studied in the enzymatic synthesis of β-lactam antibiotics by kinetically controlled N-acylation. When compared with penicillin acylase of Escherichia coli (PGA), the NPGA was significantly more efficient at syntheses of ampicillin and amoxicillin (higher S/H ratio and product accumulation) in the whole range of substrate concentrations. The degree of conversion of 6-aminopenicillanic acid to amoxicillin and ampicillin (160mM 6-APA, 350mM acyl donor methylester[Symbol: see text]HCl, pH6.3, 25°C, reaction time of 200min) with immobilized NPGA equaled 96.9% and 91.1%, respectively. The enzyme was highly thermostable with maximum activity at 60°C (pH8.0) and 65°C (pH6.0). Activity half-life at 60°C (pH8.0) and at 60°C (pH6.0) was 24min and 6.9h, respectively. Immobilized NPGA exhibited long operational stability with half-life of about 2,000 cycles for synthesis of amoxicillin at conversion conditions used in large-scale processes (230mM 6-APA, 340mM D-4-hydroxyphenylglycine methylester[Symbol: see text]HCl, 27.5°C, pH6.25). We discuss our results with literature data available for related penicillin acylases in terms of their industrial potential.

Solid-liquid equilibrium data of amoxicillin and hydroxyphenylglycine in aqueous media

The enzymatic synthesis of amoxicillin is catalyzed by Penicillin G Acylase (PGA). As byproducts, hydroxyphenylglycine and alcohol are also formed from hydrolytic reactions and antibiotic synthesis, respectively. The design of this process should be directed to promote the synthesis reaction. At the same time, it is necessary to reduce the hydrolytic reaction of amoxicillin through its crystallization or separation from the reaction medium. This work presents measurements of solid-liquid equilibrium data for amoxicillin and hydroxyphenylglycine in water at different temperatures (283.15 - 298.15 K), pH (5.5 - 7.5) and ethanol composition (0 - 70 wt.%). This information is relevant to determine the conditions that offer the lowest solubility for the antibiotic, favoring its separation and purification. All solubility data were obtained using an analytical method with indirect determination by UV spectroscopy. Ideal thermodynamic modeling was applied to describe the experimental solubility data sets.

Assessing the enzymatic synthesis of amoxicillin using different ionic liquids

Assessing the enzymatic synthesis of amoxicillin using different ionic liquids

Enzymatic synthesis of amoxicillin by penicillin G acylase in the presence of ionic liquids

A major obstacle for the industrial implementation of the enzymatic synthesis of β-lactam antibiotics is the limited yield of the product, due to undesirable hydrolytic reactions. This drawback can be partially avoided by reducing the water activity in the medium. Ionic liquids (ILs) have emerged as an alternative to conventional organic media due to their high thermal and chemical stability, negligible vapor pressure, non-flammability, and easy recycling. In this context, this paper assesses the catalytic activity of penicillin G acylase (E.C.3.5.1.11) in the synthesis of amoxicillin using different ILs, all based on the 1-butyl-3-methylimidazolium cation. An increase of 400% in selectivity (synthesis/hydrolysis, S/H ratio) was observed for the reactions carried out with BMI·PF6 as a cosolvent at 75% (vIL/vwater) when compared to the totally aqueous medium (using phosphate buffer). This figure reached 350% for BMI·NTf2, while for BMI·BF4 there was only a slight increase in selectivity. The highest conversion of the β-lactam nucleus (6-APA) was achieved using BMI·NTf2 as a cosolvent at 71% (v/v), more than 36% above the one in water. No deactivation of the enzyme after the reactions was observed in any of the ILs, and the physical integrity of the biocatalyst particles was maintained.

Facile one-pot synthesis of gold nanoparticles and their sensing protocol

This communication reports facile one pot synthesis of amoxicillin and sodium salt of amoxicillin stabilized gold nanoparticles (Au-NPs). Primarily the cyclic thioether linkage i.e. the thiozolidine ring of amoxicillin is utilized for sequestering Au(III). Fluorescence quenching of these clusters makes it an efficient protocol for sensing Cu(2+) at nano scale levels.

One-pot, two-step enzymatic synthesis of amoxicillin by complexing with Zn2+

A one-pot, two-step enzymatic synthesis of amoxicillin from penicillin G, using penicillin acylase, is presented. Immobilized penicillin acylase from Kluyvera citrophila was selected as the biocatalyst for its good pH stability and selectivity. Hydrolysis of penicillin G and synthesis of amoxicillin from the 6-aminopenicillanic acid formed and D-p-hydroxyphenylglycine methyl ester were catalyzed in situ by a single enzyme. Zinc ions can react with amoxicillin to form complexes, and the yield of 76.5% was obtained after optimization. In the combined one-pot synthesis process, zinc sulfate was added to remove produced amoxicillin as complex for shifting the equilibrium to the product in the second step. By controlling the conditions in two separated steps, the conversion of the first and second step was 93.8% and 76.2%, respectively. With one-pot continuous procedure, a 71.5% amoxicillin yield using penicillin G was obtained.

Immobilization of penicillin G acylase onto amino-modified silica hydrogel

Amino-modified silica hydrogel (N-MSHG) was prepared by a simple sol-gel processing via the co-condensation of commercial silica sol with 3-aminopropyltrieoxysilane. Penicillin G acylase (PGA), a model enzyme, was covalently immobilized onto the N-MSHG and then was used for the enzymatic synthesis of amoxicillin. The samples were characterized by Nitrogen sorption analysis, FT-IR and thermal gravimetric analysis (TGA). The results showed that the amino-modified gel was a mesoporous material with an average pore size of 12.64±0.17 nm. The immobilization process was efficient and the immobilized enzyme showed high catalytic efficiency. The yield of the synthesis of amoxicillin in aqueous media was 38% for 2.5 h. This sol-gel preparation is simple and shows prominent potential value in industrial processing.

Enzymatic Synthesis of Amoxicillin via a One-pot Enzymatic Hydrolysis and Condensation Cascade Process in the Presence of Organic Co-solvents

A cascade reaction combining the enzymatic hydrolysis of Penicillin G potassium salt (PGK) with the kinetically controlled enzymatic coupling of in situ formed 6-aminopenicillanic acid (6-APA) with p-hydroxyphenylglycine methyl ester (D-HPGM) to give amoxicillin as the final product by using a single enzyme has been demonstrated successfully. Ethylene glycol (EG) was employed as a component of reaction buffer to improve the synthesis yield. Reaction parameters, including different cosolvents, EG content, the loading of immobilized penicillin G acylase (IPA), and reaction temperature and time were studied to evaluate their effects on the reaction. The best result of 55.2% yield was obtained from the reaction which was carried out in the mixed media containing 40% sodium dihydrogen phosphate buffer (apparent pH 6.0) and 60% EG (v/v), with the initial concentration 150 mM and 450 mM of PGK and D-HPGM, respectively, catalyzed by 50 IU/mL IPA at 25 degrees C for 10 h. The IPA could be recycled for nine batches without obviously losing of catalytic activity. The important strategy will have potential application in the beta-lactam antibiotics industry due to the advantages of saving the effort of isolating 6-APA, reducing usual enzymatic steps and the industrial cost of amoxicillin synthesis.

Self‐renaturing enzymes: Design of an enzyme‐chaperone chimera as a new approach to enzyme stabilization

Molecular chaperones in aqueous-organic mixtures can broaden the utility of biocatalysis by stabilizing enzymes in denaturing conditions. We have designed a self-renaturing enzyme-chaperone chimera consisting of penicillin amidase and a thermophilic chaperonin that functions in aqueous-organic mixtures. The flexible linker separating the enzyme and chaperone domains was optimized and the design was extended to incorporate a chitin binding domain to facilitate immobilization of the chimera to a chitin support. The initial specific activity of penicillin amidase was not compromised by the enzyme-chaperone fusion or by immobilization. The total turnover number of immobilized chimera for amoxicillin synthesis in aqueous-methanol mixtures was 2.8 times higher after 95 h than the total turnover number of the immobilized penicillin amidase lacking a chaperone domain. Similarly, in 32% methanol the soluble chimera was active for over three times longer than the enzyme alone. This approach could easily be extended to other enzyme systems.

Redirecting the inactivation pathway of penicillin amidase and increasing amoxicillin production via a thermophilic molecular chaperone

We have previously shown that a single-subunit thermosome from Methanocaldococcus jannaschii (rTHS) can stabilize enzymes in semi-aqueous media (Bergeron et al., 2008b). In the present study, rTHS was used to stabilize penicillin amidase (PGA) in methanol-water mixtures. Including methanol in the reaction medium for amoxicillin synthesis can suppress unwanted hydrolysis reactions but inactivate PGA. Inactivation and reactivation pathways proposed for PGA illustrate the predictability of enzyme stabilization by rTHS in co-solvents. Calcium was necessary for reversible dissociation of the two PGA subunits in methanol-water and the presence of calcium resulted in an enhancement of chaperone-assisted stabilization. rTHS also acted as a stabilizer in the enzymatic synthesis of the beta-lactam antibiotic amoxicillin. rTHS stabilized PGA, increasing its half-life in 35% methanol by fivefold at 37 degrees C. Stabilization by rTHS was enhanced but did not require the presence of ATP. Including rTHS in fed-batch reactions performed in methanol-water resulted in nearly 4 times more amoxicillin than when the reaction was run without rTHS, and over threefold higher selectivity towards amoxicillin synthesis compared to aqueous conditions without rTHS. The thermosome and other thermophilic chaperones may thus be generally useful for stabilizing enzymes in their soluble form and expanding the range of conditions suitable for biocatalysis.

Anhydrous tert-pentanol as a novel media for the efficient enzymatic synthesis of amoxicillin

The efficient enzymatic synthesis of amoxicillin using anhydrous tert-pentanol as a novel media has been demonstrated for the first time. p-OH-Phenylglycine methyl ester (HPGM) was selected as the activated acyl donor due to its good solubility in organic solvents. The screening results of 21 organic solvents showed that solvents with either strong polarity or poor substrate solubility were unfavorable. Remarkable catalytic activity of the immobilized penicillin acylase (IPA) from Escherichia coli was retained in tert-pentanol, and high yield could be obtained. Effects of various parameters such as acyl donor, water content or cosolvents of tert-pentanol, substrate concentration, temperature, etc., on the enzymatic synthesis of amoxicillin in tert-pentanol were investigated systematically. The best reaction medium, the optimal temperature, initial concentration of 6-APA and HPGM and concentration of enzyme were tert-pentanol, 15 °C, 100, 200 mM and 20 IU/mL, respectively. Under the optimal conditions, the yield of amoxicillin was as high as 88% after a reaction time of 20 h.