- Evaluation of different glutaryl acylase mutants to improve the hydolysis of cephalosporin C. in the absence of hydrogen peroxide
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2-Oxoadipoyl-7-ACA is an intermediate in the conversion of cephalosporin C. (CPC) to 7-aminocephalosporanic acid (7-ACA) when using a new route involving D-amino acid oxidase, catalase and glutaryl acylase. A key point in the reaction design is to avoid the accumulation of hydrogen peroxide in the reaction medium as the yields of 7-ACA decrease in the presence of this compound due to its low stability. Looking for an enzyme with improved activity towards 2-oxoadipoyl-7-ACA, different mutants of glutaryl acylase from Pseudomonas SY-77 with an improved activity towards adipoyl-7-ACA were evaluated. The best results on 2-oxoadipoyl-7-ACA hydrolysis were found with the double mutant Y178F+F375H, which showed a Kcat increase of 6.5-fold and a K m decrease of 3-fold compared to the wild-type (wt) enzyme. When this enzyme was tested in the tri-enzymatic system to convert CPC into 7-ACA, this mutant permitted us to reach more than an 80% yield of 7-ACA using a 3-fold mass excess compared to DAAO; while the wt enzyme gave only a 40% yield. Therefore, the application of this new mutant to the one-pot conversion of CPC to 7-ACA gives very good result in terms of efficiency, yield and rate of the process.
- Lopez-Gallego, Fernando,Betancor, Lorena,Sio, Charles F.,Reis, Carlos R.,Jimenez, Pol Nadal,Guisan, Jose M.,Quax, Wim J.,Fernandez-Lafuente, Roberto
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Read Online
- Reversible immobilization of cephalosporin C acylase on epoxy supports coated with polyethyleneimine
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In this study, a recombinant cephalosporin C acylase (CCA) was covalently or physically immobilized on an epoxy-activated support LX1000-EPC4 (EP) or its derivatives, EP-polyethyleneimine (EP-PEI) and EP-ethylenediamine (EP-EDA) with cationic groups on the surface. Zeta potential was used as a tool for activated carrier analysis and immobilization analysis. The EP-PEI (the cationic polymer PEI grafted support) showed higher zeta potential than EP-EDA (the small diamine EDA modified support) and EP support. Among these three supports, immobilization of CCA on EP-PEI had the highest specific activity according to the range of enzyme loadings. Michaelis constant Km values of EP-PEI-CCA and EP-EDA-CCA were 22 mM and 30 mM, respectively, which were lower than that of the free enzyme (43 mM), suggesting that the supports zeta potential is related to the affinity of the enzyme for the substrate. The enzyme immobilized on EP-PEI showed a much higher thermal stability (stabilization factor of 32-fold compared with the free enzyme) than that on the EP-EDA (stabilization factor of 5.5-fold) and EP supports (stabilization factor of 1.7-fold). The adsorption of CCA on EP-PEI support was very strong and reversible. The CCA could be thoroughly desorbed using a high concentration of NaCl (e.g., 2 M) at low pH value (pH 3.0). The regenerated EP-PEI support could then be reused for enzyme immobilization.
- Wei, Yanmei,Luo, Hui,Chang, Yanhong,Yu, Huimin,Shen, Zhongyao
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Read Online
- On the substrate preference of glutaryl acylases
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The substrate preferences of three acylases - two wild-type enzymes and an evolved variant obtained by directed evolution - which are prototypical enzymes for glutaryl-7-ACA acylase and cephalosporin C acylase subfamilies, have been investigated. A preliminary screening of enzymes' performances on a large set of substrates has been carried out by a colorimetric assay performed in 96-well plates and by a pH-Stat monitoring the hydrolytic activities. Subsequently, kinetic data for selected substrates have been determined, thus elucidating the substrate preference of members of glutaryl-7-ACA acylase vs. cephalosporin C acylase subfamilies. These achievements pave the way to the ability of choosing the best enzyme for the hydrolysis of different compounds of industrial importance.
- Rosini, Elena,Monelli, Claudia Stella,Pollegioni, Loredano,Riva, Sergio,Monti, Daniela
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experimental part
p. 52 - 58
(2012/04/11)
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- One-pot conversion of cephalosporin C to 7-aminocephalosporanic acid in the absence of hydrogen peroxide
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The main drawback in the production of 7-aminocephalosporanic acid (7-ACA) at the industrial level is the inactivation of the enzymes implicated in the process due to the presence of hydrogen peroxide during the reaction. As an alternative, we have developed the conversion of cephalosporin C to 7-ACA in a single reactor without the presence of hydrogen peroxide during the reaction, achieving more than 80% yield. In order to develop this process, D-amino acid oxidase (DAAO) was co-immobilized with catalase (CAT), which is able to fully eliminate in situ the hydrogen peroxide formed by the neighbouring DAAO molecules. Thus, the product of the reaction is only α-ketoadipyl-7-ACA. This system prevents the inactivation of the oxidase by hydrogen peroxide, solving the main problem of the enzymatic process. Moreover, we have found that α-ketoadipyl-7-ACA is recognized as a substrate by glutaryl acylase (GAC) and hydrolyzed as long as glutaric acid is absent from the reaction medium (because it is able to inhibit the hydrolysis). The low stability of α-ketoadipyl-7-ACA justifies the use of a single reactor, in which glutaryl acylase is already present when this substrate is generated. Thus, the whole process may (and must) be performed in a single step, and in the absence of hydrogen peroxide that could affect the stabilities of the involved enzymes.
- Lopez-Gallego, Fernando,Batencor, Lorena,Hidalgo, Aurelio,Mateo, Cesar,Fernandez-Lafuente, Roberto,Guisan, Jose M.
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p. 1804 - 1810
(2007/10/03)
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- Influence of substrate structure on PGA-catalyzed acylations. Evaluation of different approaches for the enzymatic synthesis of cefonicid
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The influence of the substrate structure on the catalytic properties of penicillin G acylase (PGA) from Escherichia coli in kinetically controlled acylations has been studied. In particular, the affinity of different β-lactam nuclei towards the active site has been evaluated considering the ratio between the rate of synthesis (vs) and the rate of hydrolysis of the acylating ester (vhl). 7-Aminocephalosporanic acid (7-ACA) and 7-amino-3-(1-sulfomethyl-1,2,3,4-tetrazol-5-yl)thiomethyl-3-cephem-4-carboxylic acid (7-SACA) showed a good affinity for the active centre of PGA. The enzymatic acylation of these nuclei with R-methyl mandelate has been studied in order to evaluate different approaches for the enzymatic synthesis of cefonicid. The best results have been obtained in the acylation of 7-SACA. Cefonicid (8) was recovered from the reaction mixture as the disodium salt in 65% yield and about 95% of purity. Furthermore, through acylation of 7-ACA, a "one-pot" chemo-enzymatic synthesis was carried out starting from cephalosporin C using three enzymes in sequence: D-amino acid oxidase (DAO), glutaryl acylase (GA) and PGA. Cefonicid disodium salt was obtained in three steps, avoiding any intermediate purification, in 35% overall yield and about 94% purity. This approach presents several advantages compared with the classical chemical processes.
- Terreni, Marco,Tchamkam, Joseph Gapesie,Sarnataro, Umberto,Rocchietti, Silvia,Fernandez-Lafuente, Roberto,Guisan, Jose M.
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p. 121 - 128
(2007/10/03)
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- Glutaryl Acylases: One-Reaction Enzymes or Versatile Enantioselective Biocatalysts?
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A significant broad substrate specificity, that crosses over the usual β-lactam derivatives, has been observed with an industrial glutaryl-7-aminocephalosporanic acid acylase (GA). This enzyme possesses significant enantioselective amidase and even esterase activity, with a stereopreference for the S-enantiomer. The easy separation of products from unreacted reagents, possessing different physical-chemical properties, is achieved by solvent extraction, avoiding chromatography or distillation during reaction work-up.
- Raimondi, Stefano,Monti, Daniela,Pagnoni, Ugo Maria,Riva, Sergio
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p. 783 - 789
(2007/10/03)
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- Dimethyl formiminium chloride chlorosulphate derivatives useful as intermediates for producing cephalosporins
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This invention relates to reactive derivatives of 2-(2-aminothiazol-4-yl)-2-methoxyimino acetic acid and 1H-tetrazol-1-acetic acid of the following general formula I, STR1 wherein R3 = STR2 as well as to use thereof in the manufacture of cephalosporin antibiotics such as cefotaxime, ceftriaxone and cefazolin.
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- One-pot chemoenzymatic synthesis of 3'-functionalized cephalosphorines (cefazolin) by three consecutive biotransformations in fully aqueous medium
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We illustrate a new chemoenzymatic synthesis of cefazolin from cephalosporin C, involving three consecutive biotransformations in full aqueous medium. This one-pot three-step synthesis includes the D-amino acid oxidase catalyzed oxidative deamination of the cephalosporin C side chain, hydrolysis of the resulting glutaryl derivative catalyzed by glutaryl acylase, and the final penicillin G acylase (PGA)-catalyzed acylation of 7- aminocephalosporanic acid (1, 7-ACA). The product, 7-[(1H-tetrazol-1- yl)acetamido]-3-(acetoxymethyl)-Δ3-cephem-4-carboxylic acid (5), was used as an intermediate for cefazolin synthesis by 3'-acetoxy group displacement with 2-mercapto-5-methyl-1,3,4-thiadiazole. Very high yields have been achieved with all the enzymatic reactions performed; high product concentrations were obtained in short reaction times. This synthetic approach presents several advantages when compared with the conventional chemical processes. The use of the toxic reagents and chlorinated solvents is avoided, while the substrate specificity and chemoselectivity of the enzymes makes reactive group protection and intermediate purification unnecessary. The enzymatic deacylation of cephalosporin C was performed by the simultaneous use of D-amino acid oxidase and glutaryl acyclase. The substrate specificity of PGA allowed the acylation of 7-ACA (1) to be performed without purification from the glutaric acid produced during the enzymatic deacylation. These results were achieved by optimization and correct assembly of the different biotransformations involved. Special attention has been applied to the kinetically controlled acylation reaction. High yields were obtained through a careful selection of the enzyme catalyst, experimental conditions, and synthetic strategy.
- Justiz,Fernandez-Lafuente,Guisan
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p. 9099 - 9106
(2007/10/03)
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- Chemoenzymatic one-pot synthesis of cefazolin from cephalosporin C in fully aqueous medium, involving three consecutive biotransformations catalyzed by D-aminoacid oxidase, glutaryl acylase and penicillin G acylase
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A new chemoenzymatic synthesis of Cefazolin through the correct assembly of three biotransformations catalyscd by D-aminoacid oxidase, glutaryl acylase and penicillin G acylase is described. This multienzymatic synthesis has been performed from the natural Cephalosporin C in fully aqueous medium without intermediate purification stages. Almost quantitative yields have been achieved in all the enzymatic reactions.
- Fernandez-Lafuente, Roberto,Guisan, Jose M.,Pregnolato, Massimo,Terreni, Marco
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p. 4693 - 4696
(2007/10/03)
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- Method for manufacture of cephalosporins and intermediates thereof
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This invention relates to reactive derivatives of 2-(2-aminothiazol-4-yl)-2-methoxyimino acetic acid and 1H-tetrazol-1-acetic acid of the following general formula I, whereinR3= as well as to use thereof in the manufacture of cephalosporin antibiotics such as cefotaxime, ceftriaxone and cefazolin.
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- Preparation and use of 7-[(2-carboalkoxy-1-methylethenyl)amino]-3-hydroxymethyl-3-cephem-4-carboxylic acids
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This invention relates to a method of producing 3-alkanoyloxymethyl-3-cephem-4-carboxylic acids from 3-hydroxymethyl-3-cephem-4-carboxylic acids in an aqueous medium for a practical, large-scale production. Moreover, the invention provides an ideal intermediate for the process.
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- Process for the preparation of derivative of 7-[(2-hydroxyimino)-acetamido]-cephalosporanic acid
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A novel process for the preparation of syn isomers of cephalosporanic acid derivatives of the formula STR1 comprising reacting first in a solvent and optionally in the presence of a base, a compound of the formula STR2 with a compound of the formula wherein R4 is selected from the group consisting of optionally substituted alkyl, aryl and aralkyl and Hal is a halogen and reacting the resulting product in a solvent and optionally in the presence of a base with a compound of the formula STR3 to obtain the compound of formula I' which are known to possess good antibiotic properties.
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- Mechanism of β-lactam ring opening in cephalosporins
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The mechanism of the aminolysis of cephalosporins is a stepwise process. A tetrahedral intermediate is formed by the reversible addition of the amine to the beta -lactam carbonyl carbon. Expulsion of the attacking amine from the tetrahedral intermediate occurs faster than fission of the beta -lactam C-N bond. The reaction proceeds by trapping the intermediate with base. Expulsion of a leaving group at C-3 prime in cephalosporins is not concerted with nucleophilic attack of the amine on the beta -lactam carbonyl carbon and makes little difference to the rate of beta -lactam C-N bond fission.
- Page,Proctor
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p. 3820 - 3825
(2007/10/02)
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- The deblocking of cephalosporin benzhydryl esters with formic acid
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The synthesis of benzhydryl esters of cephalosporin derivatives (4-6) and removal of the benzhydryl protecting group with formic acid are described.
- Kametani,Sekine,Honda
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p. 4545 - 4547
(2007/10/02)
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- Cephalosporin derivatives and process
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A novel compound selected from the group consisting of the compound of the formula STR1 the metal salts, the ammonium salt and the salts with nitrogen bases, a process for the preparation of the said compounds and a process for the preparation of 7-aminocephalosporanic acid starting from a compound of formula I.
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- Oxyacetic acid compounds and process for their manufacture
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α-Hydroxy-2-oxo-1-azetidinemethane-carboxylic acid compounds of formula STR1 wherein R1 represents a hydrogen atom or the organic residue of an alcohol, R2 represents a hydrogen atom or an acyl residue, R3 represents an organic residue and R4 represents a hydrogen atom, when R2 stands for an acyl residue, or the two groups R3 and R4 together represent a disubstituted carbon atom, when R2 stands for a hydrogen atom or an acyl group, are useful as intermediates for the manufacture of pharmacologically active compounds.
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- 7-[5-N-(n-Butoxyethoxy carbonyl and 2-chloroethoxy carbonyl)-amino] cephalosporins C
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New and useful cephem compounds derived from cephalosporin C are provided by this invention, which are valuable as intermediate for the synthesis of another bacteriocidally active cephem derivatives and which are readily extractable from its aqueous solution by means of an organic solvent and enable the cephalosporin C content in the aqueous fermentation broth filtrate to be recovered at an improved yield. Cephalosporin C present in the fermentation broth may be converted into the cephem compounds of this invention by reacting with a substituted chloroformate compound within said filtrate, and the cephem compounds so formed may then be extracted therefrom with an organic solvent. The cephem compounds of this invention further may be converted into a 7-acylamidocephalosporanic acid or 7-aminocephalosporanic acid through some successive reactions.
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