Showing papers on "Penicillin amidase published in 2003"

Biotechnological applications of penicillin acylases: state-of-the-art.

Abstract: This review describes the most recent developments in the biotechnological applications of penicillin acylases. This group of enzymes is involved mainly in the industrial production of 6-aminopenicillanic acid and the synthesis of semisynthetic β-lactam antibiotics. In addition, penicillin acylases can also be employed in other useful biotransformations, such as peptide synthesis and the resolution of racemic mixtures of chiral compounds. Particular emphasis is placed on advances in detection of new enzyme specificities towards other natural penicillins, enzyme immobilization, and optimization of enzyme-catalyzed hydrolysis and synthesis in the presence of organic solvents.

Enantioselective hydrolysis of some 2-aryloxyalkanoic acid methyl esters and isosteric analogues using a penicillin G acylase-based HPLC monolithic silica column.

Abstract: A technique based on liquid chromatography has been developed to facilitate studies of enantioselectivity in penicillin G acylase (PGA)-catalyzed hydrolysis of some 2-aryloxyalkanoic acid methyl esters and isosteric analogues PGA was covalently immobilized on an aminopropyl monolithic silica support to create an immobilized HPLC-enzyme reactor Two sets of experimental data were drawn to calculate the enantioselectivity (E) of the kinetically controlled enantiomer-differentiating reaction, the degree of substrate conversion and the enantiomeric excess of the product The developed enzymatic reactor was coupled through a switching valve to an achiral analytical column for separation and quantitation of the hydrolysis products The enantiomeric excess was determined off-line on a PGA-chiral stationary phase In this way, highly precise E values were determined A computational study related to the hydrolysis of the considered racemic esters was also carried out in order to unambiguously clarify both the su

Kinetics of enzyme acylation and deacylation in the penicillin acylase-catalyzed synthesis of beta-lactam antibiotics

Abstract: Penicillin acylase catalyses the hydrolysis and synthesis of semisynthetic beta-lactam antibiotics via formation of a covalent acyl-enzyme intermediate. The kinetic and mechanistic aspects of these reactions were studied. Stopped-flow experiments with the penicillin and ampicillin analogues 2-nitro-5-phenylacetoxy-benzoic acid (NIPAOB) and d-2-nitro-5-[(phenylglycyl)amino]-benzoic acid (NIPGB) showed that the rate-limiting step in the conversion of penicillin G and ampicillin is the formation of the acyl-enzyme. The phenylacetyl- and phenylglycyl-enzymes are hydrolysed with rate constants of at least 1000 s-1 and 75 s-1, respectively. A normal solvent deuterium kinetic isotope effect (KIE) of 2 on the hydrolysis of 2-nitro-5-[(phenylacetyl)amino]-benzoic acid (NIPAB), NIPGB and NIPAOB indicated that the formation of the acyl-enzyme proceeds via a general acid-base mechanism. In agreement with such a mechanism, the proton inventory of the kcat for NIPAB showed that one proton, with a fractionation factor of 0.5, is transferred in the transition state of the rate-limiting step. The overall KIE of 2 for the kcat of NIPAOB resulted from an inverse isotope effect at low concentrations of D2O, which is overridden by a large normal isotope effect at large molar fractions of D2O. Rate measurements in the presence of glycerol indicated that the inverse isotope effect originated from the higher viscosity of D2O compared to H2O. Deacylation of the acyl-enzyme was studied by nucleophile competition and inhibition experiments. The beta-lactam compound 7-aminodesacetoxycephalosporanic acid (7-ADCA) was a better nucleophile than 6-aminopenicillanic acid, caused by a higher affinity of the enzyme for 7-ADCA and complete suppression of hydrolysis of the acyl-enzyme upon binding of 7-ADCA. By combining the results of the steady-state, presteady state and nucleophile binding experiments, values for the relevant kinetic constants for the synthesis and hydrolysis of beta-lactam antibiotics were obtained.

Isolation and characterization of a new strain of Achromobacter sp. with β-lactam antibiotic acylase activity

Abstract: A bacterial strain producing a β-lactam antibiotic acylase, able to hydrolyze ampicillin to 6-aminopenicillanic acid more efficiently than penicillin G, was isolated from soil and characterized. The isolate was identified as Achromobacter sp. using the phenotypic characteristics, composition of cellular fatty acids and 16S rRNA gene sequence. The enzyme synthesis was fully induced by phenylacetic acid (PAA) at a concentration of 2 g l−1. PAA at concentrations up to 12 g l−1 had no negative effect on the specific activity of acylase and biomass production, but slowed down the specific growth rate. Benzoic or 4-hydroxyphenylacetic acids can also induce synthesis of the enzyme. The inducers were metabolized in all cases. Acylase activity in cell-free extracts was determined with various substrates; ampicillin, cephalexin and amoxicillin were hydrolyzed 1.5- and 2-times faster than penicillin G. A high stability of acylase activity was observed over a wide range of pH (5.0–8.5) and at temperatures above 55°C.

Fragments of pro-peptide activate mature penicillin amidase of Alcaligenes faecalis.

Abstract: Penicillin amidase from Alcaligenes faecalis is a recently identified N-terminal nucleophile hydrolase, which possesses the highest specificity constant (kcat/Km) for the hydrolysis of benzylpenicillin compared with penicillin amidases from other sources. Similar to the Escherichia coli penicillin amidase, the A. faecalis penicillin amidase is maturated in vivo from an inactive precursor into the catalytically active enzyme, containing one tightly bound Ca2+ ion, via a complex post-translational autocatalytic processing with a multi-step excision of a small internal pro-peptide. The function of the pro-region is so far unknown. In vitro addition of chemically synthesized fragments of the pro-peptide to purified mature A. faecalis penicillin amidase increased its specific activity up to 2.3-fold. Mutations were used to block various steps in the proteolytic processing of the pro-peptide to obtain stable mutants with covalently attached fragments of the pro-region to their A-chains. These extensions of the A-chain raised the activity up to 2.3-fold and increased the specificity constants for benzylpenicillin hydrolysis mainly by an increase of the turnover number (kcat).

A homology model of penicillin acylase from Alcaligenes faecalis and in silico evaluation of its selectivity.

Abstract: A three-dimensional model of the relatively unknown penicillin acylase from Alcaligenes faecalis (PA-AF) was built up by means of homology modeling based on three different crystal structures of penicillin acylase from various sources. An in silico selectivity study was performed to compare this homology model to the structure of the Escherichia coli enzyme (PA-EC) in order to find any selectivity differences between the two enzymes. The program GRID was applied in combination with the principal component analysis technique to identify the regions of the active sites where the PAs potentially engage different interactions with ligands. These differences were further analyzed and confirmed by molecular docking simulations. The PA-AF homology model provided the structural basis for the explanation of the different enantioselectivities of the enzymes previously demonstrated experimentally and reported in the literature. Different substrate selectivities were also predicted for PA-AF compared to PA-EC. Since no crystallographic data are available for PA-AF to date, the three-dimensional homology model represents a useful and efficient tool for fully exploiting this attractive and efficient biocatalyst, particularly in enantioselective acylations of amines.

An enzymatic process for preparing beta-lactams

Abstract: An enzymatic process under kinetical control for preparing b-lactams, by coupling the 7-amino group of a cephalosporin nucleus with the carboxylic function of an acetic acid derivative consisting of a residue with p-electrons, a short spacer and a carboxylic function activated as methyl- or ethylester. The process comprises using a penicillin amidase or a-amino acid esterase as free enzyme or in any suitable immobilised form; applying at least 100 mmol/l cephalosporin nucleus as long as it is soluble, otherwise 50 - 100 % of its maximum solubility is charged, applying the acetic acid activated as ester in a 1 - 3 fold molar ratio, and adjusting the pH to 6.0 - 8.0 and the temperature to 0 - 30°C.

Unexpected enantioselectivity and activity of penicillin acylase in the resolution of methyl 2,2-dimethyl-1,3-dioxane-4-carboxylate

Abstract: The penicillin acylase (PA) from E. coli catalyzes the hydrolytic kinetic resolution of methyl 2,2-dimethyl-1,3-dioxane-4-carboxylate with remarkably high enantioselectivity and catalytic efficiency. This result is highly unusual as this ester does not contain the phenylacetic acid residue, normally considered to be a prerequisite for high activity and enantioselectivity in PA catalyzed resolutions. The apparent enantioselectivity (Eapp) was found to be high (>50) at neutral or slightly acidic pH and to decrease at more alkaline pH (>7.5) due to significant non-specific chemical hydrolysis. Similarly, enantioselectivity increased with decreasing temperature. The substrate concentration had only a slight effect on enantioselectivity and activity. The rate of hydrolysis of ester 1 is comparable to that for PA’s “natural” substrate, penicillin G.

Method for producing cephalexin

Abstract: The invention relates to a method for producing cephalexin with the aid of a penicillin amidase that is immobilized on a pearl-shaped, cross-linked and hydrophilic copolymer that binds to ligands having nucleophilic groups.

Immobilized penicillin amidase carrier and its preparing method

Abstract: The present invention discloses a kind of immobilized penicillin amidase carrier and its preparation process. The immobilized pencillin amidase carrier is prepared with surfactant as dispersant and vinyl compound as monomer and through crosslinking polymerization in reverse suspension technology. The carrier immobilized pencillin amidase has an apparat enzyme activity in preparing 6-APA with benzyl pencillin potassium as high as 125 IU/g. The polymer bead carrier containing epoxy group of the present invention has high apparent enzyme activity, simple technological process, easy operation andcheap material and may be used in industrial production.

Cell-Free Expression of the Heterodimeric Protein Penicillin G Amidase in a Functionally Active Form

Abstract: Penicillin G amidase (PGA, EC 3.5.1.11) from E. coli is a hydrolytic enzyme belonging to the structural superfamily termed N-terminal nucleophile (Ntm) amidohydrolase. It consists of two different subunits-alpha subunit (109 aa, 24 kDa) and beta subunit (557 aa, 62 kDa)-which are held together by non covalent forces. It does not contain any cystein or disulfide bridges. The N-terminal serine residue (Ser290) of the beta subunit constitutes the base in the catalytic cycle and acts as a nucleophile, whereas the alpha subunit mediates the substrate specificity. PGA displays a compact cone-shaped form with a single Cat2+ binding site. Cat2+-binding is presumed to be an important trigger of processing [1].

AN ENZYMATIC PROCESS FOR PREPARING β-LACTAMS

Abstract: An enzymatic process under thermodynamical control for preparing b-lactams, by coupling the 7-amino group of a cephalosporin nucleus with the carboxylic function of an acetic acid derivative consisting of a residue with p-electrons, a short spacer and a carboxylic function The process comprises using a penicillin amidase as free enzyme or in any suitable immobilised form; applying no or maximum 10 % organic solvent, applying at least 100 mmol/l cephalosporin nucleus, applying the free acetic acid derivative in a 3 - 5 fold molar ratio, reducing the pH by acid according the reaction progress from initially pH 75 - 625 to pH 625 - 50 and adjusting 10 - 40°C