Solid-liquid equilibrium of substrates and products of the enzymatic synthesis of ampicillin (original) (raw)
Direct Observation of Growth Rate Dispersion in the Enzymatic Reactive Crystallization of Ampicillin
Processes, 2019
Prediction and control of crystal size distributions, a prerequisite for production of consistent crystalline material in the pharmaceutical industry, requires knowledge of potential non-idealities of crystal growth. Ampicillin is one such medicine consumed in crystal form (ampicillin trihydrate). Typically it is assumed that all crystals of the same chemical and geometric type grow at the same rate, however a distribution of growth rates is often observed experimentally. In this study, ampicillin produced enzymatically is crystallized and a distribution of growth rates is observed as individual crystals are monitored by microscopy. Most studies of growth rate dispersion use complex flow apparatuses to maintain a constant supersaturation or imprecise measurements of size distributions to reconstruct growth rate dispersions. In this study, the controllable enzyme reaction enables the same information to be gathered from fewer, less complicated experiments. The growth rates of individ...
Effect of different variables in the solubility of ampicillin and corresponding solid phase
Fluid Phase Equilibria, 2018
Ampicillin belongs to the class of beta-lactam antibiotics, which corresponds the most prescribed antibiotics in medicine. This justifies the importance of elucidating the effect of several variables for the synthesis process (pH, temperature, co-solvent) on the behavior of the compounds involved in the enzymatic reaction. This paper aims to present new ampicillin solubility data in aqueous medium and the characterization of the solid phase of the antibiotic. In the phase equilibrium experiments it was considered the effect of pH (3 up to 7.5), temperature (283.15 K up to 298.15 K) and the ethanol concentration in the aqueous solution (varying between 0 and 70 wt% ethanol). The solubility curves deviate from U-shape solubility curves with respect to pH in the range of 0 to 70 wt%. This is due to ampicillin anhydrous becomes trihydrated molecule in the solid phase at alkaline pH region, changing the antibiotic properties significantly. This change in crystalline structure was confirmed by characterization of antibiotic solid phase carried out from thermogravimetric (TGA), Fourier Transform Infrared (FTIR) spectroscopy, Scanning Electron Microscopy (SEM) and X-ray Diffraction (XRD) analyses. Dissociation constants have also been measured at the studied values of temperature and ethanol composition, using potentiometric titration. Finally, a thermodynamic model considering the ideal solution was applied to describe mathematically the solubility curves measured close to experimental uncertainties.
Molecular Physics, 2001
The aggregation of ampicillin in water has been examined by conductivity measurements over the temperature range 288.15±313.15 K and light scattering. These measurements indicate the formation of two critical concentrations over the range 0±0.35 mol kg ¡ 1 . Aggregation number and e ective aggregate charge were calculated from the static light scattering data according to the Anacker and Westwell treatment. Thermodynamic parameters of aggregate formation were obtained from a form of the mass action model applicable to systems of low aggregation number. This method was applied at both critical concentrations. A valence of one was used for the monomers present in the ®rst equilibrium. The second equilibrium was between aggregates of two di erent sizes, in this case, the valence of the aggregates being the e ective charge calculated from the Anacker and Westwell treatment. Experimental results show that free energies of micellization for ampicillin are higher for the ®rst critical concentration and in the same range, but lower than for other penicillins. The enthalpies of micellization become negative when the temperature is increased, but the variation is three times greater for the ®rst critical concentration than for the second.
Applied Biochemistry and Biotechnology, 2002
Immobilized penicillin acylase is a moderately priced versatile enzyme, that is able to catalyze the synthesis of derived penicillins and cephalosporins from the corresponding β-lactam nuclei and proper side-chain precursors. Kinetically controlled synthesis is a better strategy when product yield is a key issue. Yield should increase at reduced water activity by depressing the competing hydrolytic reactions in favor of synthesis; therefore, organic cosolvents can be a suitable reaction media for synthesis. Using response surface methodology and product yield as objective function, temperature and pH were optimized in the kinetically controlled synthesis of ampicillin using previously screened cosolvents and reaction conditions. Optimum pH was 6.0 for ethylene glycol (EG) and glycerol (GL) and 6.6 for 1-2 propanediol (PD); optimum temperature was 30°C for GL and for EG and PD was in the lower extreme of the range studied, optimum lying below 26°C. Maximum molar yields predicted by the model were 58, 51, and 46% for EG, GL, and PD, respectively, which were experimentally validated. Highest yield in aqueous buffer was always <40%. Molar yields about 60% compare favorably with values reported for the kinetically and thermodynamically controlled synthesis of ampicillin and other derived penicillins.
Biotechnology and Bioengineering, 2001
The synthesis of ampicillin catalyzed by Escherichia coli penicillin acylase was optimized in an aqueous system with partially dissolved antibiotic nucleus 6-aminopenicillanic acid (6-APA). The yields of both 6-APA and acyl donor could be improved by repetitively adding substrates to the reaction, allowing the concentration of 6-APA to remain saturated throughout. In this reaction concept, with four subsequent additions of substrates, 97% conversion of 6-APA and 72% of D-(−)phenylglycine methyl ester (D-PGM) to ampicillin was achieved. The synthetic potential of this concept was estimated using a mathematical model which showed that by increasing the amount of added substrates a nearly quantitative conversion of 6-APA and 85% conversion of acyl donor into ampicillin could be achieved.
Enzymatic reactive crystallization for improving ampicillin synthesis
Chemical Engineering Science, 2017
Reactive crystallization can be employed to increase the selectivity of ampicillin. New pH-sensitive model predicts concentrations for non-pH-stat batch reactions. Experiments confirm model predictions of better selectivity towards ampicillin. Using Assemblase Ò selectivity is increased 50% by parallel reaction/crystallization. Yield is improved 20% over the theoretical maximum not considering crystallization.
Enzyme and Microbial Technology, 1999
For the enzymatic synthesis of the antibiotic cephalexin, an acti¨ated acyl donor is generally used as one of the () substrates kinetically controlled approach ; howe¨er, the thermodynamically controlled approach might be of interest since there is no need for acti¨ation of the acyl donor and less waste is produced. If the synthesis reaction can be combined with an effecti¨e product remo¨al step, the thermodynamic approach can be beneficial. The thermodynamically controlled synthesis of cephalexin was studied at¨arious pH¨alues, sol¨ent concentrations, and temperatures. With direct synthesis in water, only small amounts of cephalexin were () formed 0.1 mM from 20 mM starting material by the Xanthomonas citri enzyme. Addition of water-miscible () organic sol¨ent had a positi¨e effect on synthesis by the Escherichia coli enzyme ; the equilibrium () concentration of cephalexin, howe¨er, was at best increased by a factor of 2᎐3 in methanol and triglyme. The equilibrium antibiotic concentrations reported in this study were notably lower than the¨alues reported in the literature. These differences originate from the impro¨ed analytical methods that are a¨ailable nowadays. Low product concentrations were also found for other side-chains with an amino group at the ␣-position. Side-chains without this group can be coupled and gi¨e acceptable product concentrations. For these antibiotics, a thermodynamically controlled process may be an alternati¨e to kinetically controlled coupling.
Journal of Molecular Catalysis B-enzymatic, 2001
Penicillin acylase (PA) is used in the industrial production of 6-amino penicillanic acid (6-APA). However, by proper control of reaction medium, the enzyme can be used in the reverse synthesis of β-lactam antibiotics from the corresponding β-lactam nuclei and suitable acyl donors. Under thermodynamically controlled strategy, the use of organic cosolvents can favor synthesis over hydrolysis by lowering water activity and favoring the non-ionic reactive species. Under kinetically controlled strategy using activated acyl donors, organic solvents can favor synthesis by depressing hydrolytic reactions. Results are presented on the synthesis of ampicillin from phenylglycine methyl ester and 6-APA with immobilized Escherichia coli PA in the presence of organic cosolvents. Several solvents were tested in terms of enzyme stability and solubility of substrates. Ethylene glycol, glycerol, 1–2 propanediol and 1–3 butanediol were selected accordingly and ampicillin synthesis was performed in al...
Effect of pH in the synthesis of ampicillin by penicillin acylase
Enzyme and microbial …, 1996
Recombinant Escherichia coli cells with high penicillin acylase (PA) activity were immobilized by gel entrapment with agar. This biocatalyst was used to study the effect of pH on the synthesis of ampicillin from phenylglycine methylester (PGME) and 6-aminopenicillanic acid ...
In Vivo Investigation of the Interaction of Ampicillin.pdf
In Vivo, Stability Constant, Ampicillin, Oxytetracycline and 2-Mercaptoimidazol he interaction of antibiotic Interaction of Ampicillin, Oxytetracycline and 2-Mercaptoimidazol with some Salts has been investigated by In vivo studies. It's found that there were no significant changes observed in the mean value of serum ionized calcium of the treated rats compared with the corresponding levels of control untreated rat group upon treatment with 1.4 mg/kg body weight. This is an interesting result because calcium complex ion in aqueous solutions with AMP, MI and OTC, but the same investigated compounds doesn't show significant decrease in serum ionized calcium and Sodium due to the ability of the compounds to form complexes with calcium and Sodium at physiological conditions i.e. (inside living organisms).So administration of those compounds has no side effect on serum ionized calcium on developing any hypocalcaemia related diseases.
Process Biochemistry, 2006
The effect of temperature was studied for the kinetically controlled synthesis of cephalexin and ampicillin with penicillin acylase immobilised in glyoxyl agarose. Yield increased at low temperatures in the absence and presence of ethylene glycol, while the initial ratio of synthesis to hydrolysis decreased. Arrhenius equations were used to describe the temperature dependency of the hydrolysis and synthesis rates. The effect of ethylene glycol was stronger over the yield of synthesis of cephalexin than ampicillin. In the case of cephalexin, yield increased from 82.8% in aqueous buffer to 97.6% in 50% (v/v) ethylene glycol medium at 0 8C, while at 20 8C an increase from 68.8% to 78.7% was obtained. The presence of ethylene glycol produced a greater increase in the energies of activation of the hydrolysis reactions than of the synthesis reactions, which explains the higher conversion yields obtained in the presence of the cosolvent, both for cephalexin and ampicillin. Cephalexin synthesis was optimized using an experimental design based on surface of response methodology.
Brazilian Archives of Biology and …, 2005
In this work, optimal control techniques were used to optimize the feed of reactants during the enzymatic synthesis of ampicillin in a semi-batch reactor. Simulation results showed that a semi-batch integrated reactor (with product crystallization) might achieve 88% 6-APA (6-aminepenicillanic acid) conversion and 92% of PGME (phenylglycine methyl ester) yield, with a productivity between 3.5 and 5.5 mM min-1.
Journal of Molecular Catalysis B: Enzymatic, 2000
Penicillin acylase-catalyzed ampicillin synthesis via acyl group transfer in aqueous solution is highly dependent on the Ž . initial substrate concentration. The solubility of one substrate, 6-aminopenicillanic acid 6-APA , can be advantageously enhanced by the presence of acyl donor, the second substrate. Furthermore, a comparison of enzymatic synthesis in homogeneous solution with synthesis in a heterogeneous system having partially undissolved reactants, reveals major advantages for the latter approach. In this ''aqueous solution-precipitate'' system, accumulation of both products, ampicillin Ž . and D-y -phenylglycine, proceeds through the formation of their supersaturated solutions. Subsequent precipitation of the product ampicillin positively influences the efficiency of the biocatalytic process. As a result, ampicillin synthesis proceeds Ž . in 93% conversion on 6-APA and in 60% conversion on D-y -phenylglycine methyl ester. q
Biotechnology and Bioengineering, 2002
The penicillin acylase-catalyzed synthesis of ampicillin by acyl transfer from D-(±)-phenylglycine amide (D-PGA) to 6-aminopenicillanic acid (6-APA) becomes more effective when a judiciously chosen pH gradient is applied in the course of the process. This reaction concept is based on two experimental observations: 1) The ratio of the initial synthesis and hydrolysis rates (V S /V H ) is pH-dependent and exhibits a maximum at pH 6.5±7.0 for a saturated solution of 6-APA; 2) at a ®xed 6-APA concentration below saturation, V S /V H increases with decreasing pH. Optimum synthetic ef®ciency could, therefore, be achieved by starting with a concentrated 6-APA solution at pH 7 and gradually decreasing the pH to 6.3 in the course of 6-APA consumption. A conversion of 96% of 6-APA and 71% of D-PGA into ampicillin was accomplished in an optimized procedure, which signi®cantly exceeds the ef®ciency of enzymatic synthesis performed at a constant pH of either 7.0 or 6.3. ã 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 589±593, 2002.
Feasibility of the thermodynamically controlled synthesis of amoxicillin
Journal of Molecular Catalysis B: Enzymatic, 1998
The enzymatic, thermodynamically controlled synthesis of amoxicillin in aqueous solution was measured in order to study the feasibility of a 'solid-to-solid' conversion. In aqueous solution, however, the synthetic yield of amoxicillin remains lower than the amoxicillin solubility. Therefore, a 'solid-to-solid' synthesis of amoxicillin in aqueous solution is not feasible. Synthetic yields in enzymatic condensation reactions can often be improved by adding organic solvents in monophasic systems. Addition of cosolvents is calculated to improve the apparent equilibrium constant of amoxicillin synthesis considerably, but probably not the synthetic yield, due to solubility restrictions of the reactants. q
Reaction heat measurements in the enzymatic hydrolysis reaction of penicillin GK to obtain 6-APA
Enzyme and Microbial Technology, 1996
7.5 are the penicillin hydrolysis, AHhydro,, at 35.9 f 5.7 kJ mol-' and phenyl acetic acid neutralization, AH,,,,,, at -47.1 + 3.8 kl mol-'. 0 1996 by Elsevier Science Inc.
Penicillin Acylase Catalysed Synthesis of Ampicillin in Hydrophilic Organic Solvents
Advanced Synthesis & Catalysis, 2003
Penicillin acylase (EC 3.5.1.11) from Alcaligenes faecalis, immobilised as a cross-linked enzyme aggregate (CLEA), catalysed the synthesis of ampicillin in water-miscible organic solvents at low water concentrations. Below 4% water (v/v) no reaction was observed, showing the crucial role of water in maintaining the activity of penicillin acylase. The initial value of S/H was strongly affected by the nature of the solvent, but the effect of the water content was slight in the studied range of 4 to 15%. A reaction in acetonitrile containing 8% water afforded ampicillin in up to 86% yield.