Penicillin acylase-catalyzed synthesis of ampicillin in “aqueous solution–precipitate” systems. High substrate concentration and supersaturation effect

Youshko, Maxim I; Langen, Luuk M. van; Vroom, Erik de; Moody, Harold M.; Rantwijk, Fred van; Sheldon, Roger A.; Švedas, Vytas K.

doi:10.1016/s1381-1177(00)00091-6

Cited by 58 publications

(30 citation statements)

References 25 publications

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“…Ampicillin synthesis using the obtained mutants can likely be further improved by a rational biocatalytic process design using different optimization approaches described in the literature: medium engineering (Rosell et al, 1998;Park et al, 2000;Youshko et al, 2002b;Ferreira et al, 2004), increasing the initial concentrations of acyl donor (PGA) and penicillin nucleus (6-APA) (Youshko et al, 2000), repetitive addition of substrates to the reaction mixture (Youshko et al, 2001), adequate immobilization of the enzyme (Mateo et al, 2002;Kallenberg et al, 2005) or removal of product in two-phase systems (HernandezJustiz et al, 1998;Terreni et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

“…In the second step, 6-APA is coupled to a phenylglycine side group, yielding ampicillin. This second step can also be catalyzed by PA if an ester or amide of phenylglycine is used (Bruggink et al, 1998;Kasche, 1986;Alkema et al, 2002a;Youshko et al, 2000). However, the synthetic capacities of known PAs are only modest and need to be improved for economically competitive usage in large-scale production of penicillins and cephalosporins.…”

Section: Introductionmentioning

confidence: 99%

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Saturation mutagenesis reveals the importance of residues αR145 and αF146 of penicillin acylase in the synthesis of β-lactam antibiotics

Jager

Шаповалова

Jekel

et al. 2008

Journal of Biotechnology

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Saturation mutagenesis reveals the importance of residues alpha R145 and alpha F146 of penicillin acylase in the synthesis of beta-lactam antibiotics Jager, Simon A. W.; Shapovalova, Irina V.; Jekel, Peter A.; Alkema, Wynand B. L.; Svedas, Vytas K.; Janssen, Dick B.; Švedas, Vytas K. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. AbstractPenicillin acylase (PA) from Escherichia coli can catalyze the coupling of an acyl group to penicillin-and cephalosporin-derived ␤-lactam nuclei, a conversion that can be used for the industrial synthesis of ␤-lactam antibiotics. The modest synthetic properties of the wild-type enzyme make it desirable to engineer improved mutants. Analysis of the crystal structure of PA has shown that residues ␣R145 and ␣F146 undergo extensive repositioning upon binding of large ligands to the active site, suggesting that these residues may be good targets for mutagenesis aimed at improving the catalytic performance of PA. Therefore, site-saturation mutagenesis was performed on both positions and a complete set of all 38 variants was subjected to rapid HPLC screening for improved ampicillin synthesis. Not less than 33 mutants showed improved synthesis, indicating the importance of the mutated residues in PA-catalyzed acyl transfer kinetics. In several mutants at low substrate concentrations, the maximum level of ampicillin production was increased up to 1.5-fold, and the ratio of the synthetic rate over the hydrolytic rate was increased 5-15-fold. Moreover, due to increased tendency of the acyl-enzyme intermediate to react with ␤-lactam nucleophile instead of water, mutants ␣R145G, ␣R145S and ␣R145L demonstrated an enhanced synthetic yield over wild-type PA at high substrate concentrations. This was accompanied by an increased conversion of 6-APA to ampicillin as well as a decreased undesirable hydrolysis of the acyl donor. Therefore, these mutants are interesting candidates for the enzymatic production of semi-synthetic ␤-lactam antibiotics.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Saturation mutagenesis reveals the importance of residues αR145 and αF146 of penicillin acylase in the synthesis of β-lactam antibiotics

Jager

Шаповалова

Jekel

et al. 2008

Journal of Biotechnology

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“…During the computations, the initial geometry of the molecule in the vacuum was stepwise-optimized, gradually increasing the complexity (n) Present work, (l) data from literature: (A) ampicillin (l) Rudolph et al 10 ; (B) 6-APA, (l) Rudolph et al 10 ; and (C) PG, (l) Youshko et al 5 of the theory and the bases of the molecular orbital computation. The final optimization was accomplished via the method of three hybrid parameters (B3LYP) using base 6-311, including polarization of heavy atoms.…”

Section: Solubility Of the Componentsmentioning

confidence: 99%

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

et al. 2009

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The solid-liquid equilibrium of precursors and products of the enzymatic synthesis of ampicillin (AMP) [6-aminopencillanic acid (6-APA) and D(À)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(À)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. V

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“…Among the enzymatic approaches, the kinetically controlled route shows feasibility for the synthesis of antibiotics derived from α-amino acids (Youshko et al, 2000), which uses an activated side chain donor (e.g., an ester or amide) and the penicillin nucleus, 6-aminopenicillanic acid (6-APA). In the enzymatic synthesis of amoxicillin from hydroxyphenylglycine methyl ester (HPGM) and 6-APA, as indicated in Figure 1, the Penicillin G Acylase (PGA) catalyzes both the synthesis of the antibiotic by coupling (Reaction 1) and the undesired hydrolysis of the substrate (Reaction 2) and its product (Reaction 3), generating hydroxyphenylglycine (HPG) and alcohol.…”

Section: Introductionmentioning

confidence: 99%

“…Rudolph et al (1999) presented experimental data for the solubility of ampicillin, amoxicillin and their precursors in water in a range of temperature at the isoelectric point and varying the pH at 298.15 K. The experimental data were successfully described using a simplified model proposed by Khoshkbarchi and Vera (1996). Youshko et al (2000) measured the solubility of individual components of the reaction of enzymatic synthesis of ampicillin in the pH range between 4 and 8 at 298.15 K. From these measurements, it was concluded that the precipitation of PG is almost inevitable in the course of the reaction when using high initial concentrations of substrates at any value of the pH. The optimal conditions according to these data were found to be in the range of pH between 6 and 7, the precipitation of ampicillin being effective only at pH below 7.5.…”

Section: Introductionmentioning

confidence: 99%

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

Bezerra

Chiavone‐Filho

Mattedi

2013

Braz. J. Chem. Eng.

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-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.

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Penicillin acylase-catalyzed synthesis of ampicillin in “aqueous solution–precipitate” systems. High substrate concentration and supersaturation effect

Cited by 58 publications

References 25 publications

Saturation mutagenesis reveals the importance of residues αR145 and αF146 of penicillin acylase in the synthesis of β-lactam antibiotics

Saturation mutagenesis reveals the importance of residues αR145 and αF146 of penicillin acylase in the synthesis of β-lactam antibiotics

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

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

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