Kinetics of the enzymatic synthesis of benzylpenicillin

Švedas, Vytas K.; Margolin, Alexey L.; Borisov, Ivan L.; Березин, И.В.

doi:10.1016/0141-0229(80)90103-9

Cited by 32 publications

(7 citation statements)

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“…For the preparation of semi-synthetic penicillins, the acyl donor is usually the amide or methyl ester of an aromatic carboxylic acid. In this case, the level of product accumulation is governed by the catalytic properties of the enzyme and high non-equilibrium concentrations of the acyl-transfer product can transiently be obtained (Svedas et al, 1980b;Youshko et al, 2002a,b). The ability of PA to catalyze effective acyl transfer to ␤-lactam antibiotic nuclei is very much dependent on the reaction conditions (Ferreira et al, 2004;Ospina et al, 1996;Park et al, 2000), and the type of PA and therefore can be influenced by mutating the enzyme's active site (Alkema et al, 2002b;Gabor and Janssen, 2004).…”

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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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: 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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“…For the thermodynamic equilibrium constant ( K th ), the following equation is applied K th = exp true( − Δ G° c R T true) Δ G ° c is the (pH-independent) standard Gibbs energy change.…”

Section: Model Development For Evaluation Of Process Optionsmentioning

confidence: 99%

Conceptual Process Design of Integrated Fermentation, Deacylation, and Crystallization in the Production of β-Lactam Antibiotics

Engel

Straathof

Gulik

et al. 2009

Ind. Eng. Chem. Res.

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Integration of fermentation and downstream steps is required to improve the sustainability of industrial biotechnology processes. In this context, a new integrated process for β-lactam nuclei production is proposed, which has been theoretically investigated and conceptually designed. This process is an integration of fermentation and enzymatic deacylation of adipyl-7-aminodeacetoxycephalosporanic acid (adADCA) in one reactor producing 7-aminodeacetoxycephalosporanic acid (ADCA) directly from glucose. Although the deacylation equilibrium is unfavorable at the fermentation pH, it is pulled to completion because ADCA starts to crystallize and the liberated side chain, adipic acid (AA), is consumed by the fermentation. Therefore the integrated process requires much less AA than the nonintegrated process. In addition, the new process will lead to a reduction in number of downstream processing units, should avoid the use of acids and bases for pH shifts, and might lead to a reduction in waste salts production. A conceptual process was designed including an economic and technical analysis. The design goal was to produce 2000 tons/year of ADCA of 99% purity. Rigorous simulations were performed to evaluate different process options. Among the economic advantages of the new integrated process are 13.4% lower capital investments and 7.8% lower manufacturing costs. An analogous process for 6-aminopenicillanic acid (APA) is not feasible due to chemical degradation of this product.

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“…In a later stage the accumulating amide competes increasingly with the ester as enzyme substrate and is hydrolyzed to RNH 2 and HOAc. Consequently the yield of amide increases to a maximum and then declines (Marconi et al, 1975;Svedas et al, 1980aSvedas et al, , 1980b. …”

Section: Kinetics Of Acylationsmentioning

confidence: 99%

Penicillin acylases as amidohydrolases and acyl transfer catalysts

Konecny

1981

Biotechnol Lett

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The hypothesis that an acyl-enzyme intermediate of the esterases from Xanthomonas cirri and Acetobacter turbidans is responsible for the acylation of 7-aminodeacetoxycephalosporanic acid and similar compounds by esters of amino acids, recently proposed by Kate, also accounts for the kinetics of hydrolysis of penicillin G by the amidohydrolase from E. coli and other reactions of the enzyme. Some further implications of the mechanism are derived and discussed. In recent studies concerning the acylation of 7-aminodeacetoxycephalosporanic acid (7-ADCA) by the methyl ester of D-phenyl glycine and of similar acylations catalyzed by the esterases from x. cirri and A. turbidans (Kato and coworkers 1980a, 1980b; Kate, 1980) it has been deduced that the reactions proceed via an acy!-enzyme intermediate. Kinetics of the reactions indicate a rate determining acylation of the enzyme by the ester, followed by a fast transfer of the acyl radical to the amino group of 7-ADCA or water, which compete for the acyl group as accepters. The purpose of this communication is to show that this mechanism has broader implications. In particular it accounts for * qhis paper is dedicated to Prof. G. Manecke, a pioneer of immobilized enzyme technology, with oongratulations to his 65thbirthday. 107

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Kinetics of the enzymatic synthesis of benzylpenicillin

Cited by 32 publications

References 5 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

Conceptual Process Design of Integrated Fermentation, Deacylation, and Crystallization in the Production of β-Lactam Antibiotics

Penicillin acylases as amidohydrolases and acyl transfer catalysts

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