scispace - formally typeset
Search or ask a question
Topic

Penicillin amidase

About: Penicillin amidase is a research topic. Over the lifetime, 576 publications have been published within this topic receiving 15563 citations. The topic is also known as: penicillin amidohydrolase & ampicillin acylase.


Papers
More filters
Journal ArticleDOI
TL;DR: The production of penicillin G andpenicillin V amidohydrolases or acylases was studied during the autolysis of filamentous fungi in a mineral medium, and in the same medium with phenoxyacetic acid as inducer.
Abstract: The production of penicillin G and penicillin V amidohydrolases or acylases (E.C.3.5.1.11) was studied during the autolysis of filamentous fungi in a mineral medium, and in the same medium with phenoxyacetic acid as inducer. In all the studied fungi, enzymes showing penicillin G and penicillin V amidohydrolase activities were found. Generally, an increase of these activities during fungal autolysis was observed. The presence of phenoxyacetic acid in the medium did not increase these activities. The activities found in the culture fluids were generally higher than that found in the mycelial extracts. Under these conditions, beta-lactamases (penicillinases) were not found. The fungi Alternaria alternata, Fusarium culmorum, Penicillium oxalicum, and the species Penicillium 222 were chosen to study penicillin G and penicillin V acylases. The enzymes were precipitated with tannic acid from the culture fluid of their autolyzed cultures. Some kinetic constants of these activities were determined.

12 citations

Journal ArticleDOI
TL;DR: Cloned penicillin G acylase (PGA) from Escherichia coli ATCC 11105 was mutagenized in vivo using N-methyl-N′-nitrosoguanidine to select mutants of PGA selected by their ability to allow growth of the host strain E. coli with new substrates.
Abstract: Cloned penicillin G acylase (PGA) from Escherichia coli ATCC 11105 was mutagenized in vivo using N-methyl-N′-nitrosoguanidine. Mutants of PGA were selected by their ability to allow growth of the host strain E. coli M8820 with the new substrates phenylacetyl-β-alanyl-l-proline (PhAc-βAla-Pro) phthalyl-l-leucine (Pht-Leu) or phthalylglycyl-l-proline (Pht-Gly-Pro) as sole source of proline and leucine respectively. PGA mutants were purified and immobilized onto spherical methacrylate (G-gel). The immobilized form of mutant PGA selected with (PhAc-gbAla-Pro) hydrolyzed 95% of 9 mmol penicillin G 30% faster than wild-type PGA using the same specific activities. The specific activity of the soluble enzyme was 2.7-fold, and inhibition by phenylacetic acid was halved. Immobilized PGA mutant selected with Pht-Gly-Pro hydrolyzed penicillin G 20% faster than wild-type PGA. The Km of the soluble enzyme was increased 1.7-fold. Furthermore, the latter two mutants were also 3.6-fold more stable at 45° C than wild-type PGA. The specific activity of the mutant selected with Pht-Leu was 6.3-fold lower, and inhibition by phenylacetic acid was increased 13-fold.

12 citations

Journal ArticleDOI
TL;DR: Optimization of the immobilization process led to a homogeneous distribution of the enzyme on the support surface avoiding the attachment of enzyme aggregates, and the optimal immobilized biocatalyst had a specific enzymatic activity of 26.2IUgwetcarrier−1 in the hydrolysis of penicillin V at pH 8.0 and 40°C.
Abstract: Recombinant penicillin acylase from Streptomyces lavendulae was covalently bound to epoxy-activated Sepabeads EC-EP303®. Optimization of the immobilization process led to a homogeneous distribution of the enzyme on the support surface avoiding the attachment of enzyme aggregates, as shown by confocal electron microscopy. The optimal immobilized biocatalyst had a specific enzymatic activity of 26.2IUgwetcarrier−1 in the hydrolysis of penicillin V at pH 8.0 and 40°C. This biocatalyst showed the highest activity at pH 8.5 and 65°C, 1.5 pH units lower and 5°C higher than its soluble counterpart. Substrate specificity of the derivative also showed its ability to efficiently hydrolyze other natural aliphatic penicillins such as penicillins K, F and dihydroF. The immobilized enzyme was highly stable at 40°C and pH 8.0 (t1/2=625 h vs. t1/2=397 h for the soluble enzyme), and it could be recycled for at least 30 consecutive batch reactions without loss of catalytic activity.

12 citations

Journal ArticleDOI
TL;DR: The thermosome and other thermophilic chaperones may thus be generally useful for stabilizing enzymes in their soluble form and expanding the range of conditions suitable for biocatalysis.
Abstract: We have previously shown that a single-subunit thermosome from Methanocaldococcus jannaschii (rTHS) can stabilize enzymes in semi-aqueous media (Bergeron et al., 2008b). In the present study, rTHS was used to stabilize penicillin amidase (PGA) in methanol–water mixtures. Including methanol in the reaction medium for amoxicillin synthesis can suppress unwanted hydrolysis reactions but inactivate PGA. Inactivation and reactivation pathways proposed for PGA illustrate the predictability of enzyme stabilization by rTHS in co-solvents. Calcium was necessary for reversible dissociation of the two PGA subunits in methanol–water and the presence of calcium resulted in an enhancement of chaperone-assisted stabilization. rTHS also acted as a stabilizer in the enzymatic synthesis of the β-lactam antibiotic amoxicillin. rTHS stabilized PGA, increasing its half-life in 35% methanol by fivefold at 37°C. Stabilization by rTHS was enhanced but did not require the presence of ATP. Including rTHS in fed-batch reactions performed in methanol–water resulted in nearly 4 times more amoxicillin than when the reaction was run without rTHS, and over threefold higher selectivity towards amoxicillin synthesis compared to aqueous conditions without rTHS. The thermosome and other thermophilic chaperones may thus be generally useful for stabilizing enzymes in their soluble form and expanding the range of conditions suitable for biocatalysis. Biotechnol. Bioeng. 2009;102: 417–424. © 2008 Wiley Periodicals, Inc.

12 citations

Journal ArticleDOI
TL;DR: Structural models, as well as docking analyses, can predict the positioning of penicillins G and K for catalysis and can demonstrate how binding in a productive pose is compromised when more than one bulky phenylalanine residue is introduced into the active site.
Abstract: A homologue of the Escherichia coli penicillin acylase is encoded in the genomes of several thermophiles, including in different Thermus thermophilus strains Although the natural substrate of this enzyme is not known, this acylase shows a marked preference for penicillin K over penicillin G Three-dimensional models were created in which the catalytic residues and the substrate binding pocket were identified Through rational redesign, residues were replaced to mimic the aromatic binding site of the E coli penicillin G acylase A set of enzyme variants containing between one and four amino acid replacements was generated, with altered catalytic properties in the hydrolyses of penicillins K and G The introduction of a single phenylalanine residue in position α188, α189, or β24 improved the K(m) for penicillin G between 9- and 12-fold, and the catalytic efficiency of these variants for penicillin G was improved up to 66-fold Structural models, as well as docking analyses, can predict the positioning of penicillins G and K for catalysis and can demonstrate how binding in a productive pose is compromised when more than one bulky phenylalanine residue is introduced into the active site

12 citations

Network Information
Related Topics (5)
Pseudomonas putida
6.8K papers, 230.5K citations
79% related
Immobilized enzyme
15.2K papers, 401.8K citations
78% related
Xylose
10.3K papers, 310.4K citations
77% related
Yeast
31.7K papers, 868.9K citations
77% related
Bacillus subtilis
19.6K papers, 539.4K citations
77% related
Performance
Metrics
No. of papers in the topic in previous years
YearPapers
20234
20222
20183
20175
20165
20153