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: Values for the relevant kinetic constants for the synthesis and hydrolysis of beta-lactam antibiotics were obtained and 7-aminodesacetoxycephalosporanic acid was a better nucleophile than 6-aminopenicillanic acid, caused by a higher affinity of the enzyme for 7-ADCA and complete suppression of hydrolysed of the acyl-enzyme upon binding of 7- ADCA.
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.

48 citations

Journal ArticleDOI
TL;DR: The microstructure and the catalytic properties of crosslinked enzyme aggregates of penicillin acylase (PA) obtained under different conditions were investigated in this article, where the period of time left between enzyme precipitation and the cross-linking step was found to influence the structural organization of the resulting enzyme preparation.
Abstract: The microstructure and the catalytic properties of cross-linked enzyme aggregates (CLEA) of penicillin acylase (PA) obtained under different conditions were investigated The period of time left between the enzyme precipitation and the cross-linking step was found to influence the structural organization of the resulting enzyme preparation Confocal fluorescent microscopy of the so-called “fresh” and “mature” CLEAs PA allowed to estimate the “characteristic” diameter of CLEA PA particles, which appeared to be about 16 μm, and revealed that the “mature” type was composed of relatively big particles as compared to the “fresh” type Complementary kinetic studies showed that the “mature” CLEA PA were more effective in both hydrolytic and synthetic reactions It was suggested that the aggregate size might regulate the extent of covalent modification of PA and thereby influence the catalytic properties of CLEA

48 citations

Journal ArticleDOI
TL;DR: In this paper, a composite support polyethyleneimine (PEI) was grafted onto the surface of silica gel particles via the coupling effect of γ-chloropropyl trimethoxysilane (CP), and the novel composite support PEI/SiO2 was prepared.

47 citations

Journal ArticleDOI
TL;DR: Results of these investigations indicate that the alpha peptide constitutes a folding domain and suggest that it plays a key role in folding of the precursor for penicillin acylase.
Abstract: The solution conformation properties of penicillin G acylase (EC 3.5.1.11) have been characterised by near- and far-ultraviolet circular dichroism, steady-state and time-resolved fluorescence spectroscopy and differential sedimentation velocity. The enzyme (86 kDa) was found to be spherical and stable unfolding over a narrow range of urea concentrations in an apparently cooperative fashion with a mid-point of 4.5 M urea. Separation of its constituent alpha and beta peptides (23.8 kDa and 62.2 kDa, respectively) was accompanied by loss of enzyme activity and unfolding, the kinetics of unfolding being highly dependent upon urea concentration. Urea gradient gel electrophoresis showed that the separated beta peptide aggregates over a wide range of urea concentrations but that the alpha peptide refolds reversibly to a compact state. Physical studies showed that the refolded alpha peptide has a compact but asymmetric structure with more alpha helix than the native enzyme, but is more sensitive to denaturant. The latter is suggested to be due to a hydrophobic patch detected by 8-anilino-1-naphthalene sulfonic acid binding and which is normally covered by the beta peptide in the native enzyme. The results of these investigations indicate that the alpha peptide constitutes a folding domain and suggest that it plays a key role in folding of the precursor for penicillin acylase.

47 citations

Journal ArticleDOI
TL;DR: In this paper, les deux sous-unites de la penicilline acylase d'Escherichia coli proviennent d'un precurseur commun polypeptidique localise exclusivement dans la membrane.
Abstract: Les resultats montrent que les deux sous-unites de la penicilline acylase d'Escherichia coli proviennent d'un precurseur commun polypeptidique localise exclusivement dans la membrane

47 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