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 published on a yearly basis
Papers
More filters
TL;DR: The biocatalyst was very stable under reaction conditions, so that a very high global productivity is anticipated, making the enzymatic process competitive with existing chemical synthesis.
Abstract: The kinetically controlled synthesis of cephalexin (CEX) in ethylene glycol (EG) was previously optimized at moderate substrate concentrations and high enzyme to substrate ratio, obtaining yields close to stoichiometric. However, substrate concentrations were low and enzyme loads high enough for production purpose. The synthesis of cephalexin in 40% (v/v) ethylene glycol at 20 °C and pH 7.0 with glyoxyl-agarose immobilized penicillin acylase (GAPA) was studied at high substrates concentrations to the point of saturation and beyond. Phenylglycine methyl ester (PGME) was the acyl donor at a molar ratio of 3 with respect to nucleophile. At initially homogeneous conditions with nucleophile concentration close to its solubility and at low enzyme to substrate ratio, productivity increase eight times and specific productivity five times with respect to a control at moderate substrates concentrations and high enzyme to substrate ratio. At initially heterogeneous conditions with partially undissolved nucleophile and low enzyme to substrate ratio, increases in productivity and specific productivity were eleven and seven times, respectively. The biocatalyst was very stable under reaction conditions, so that a very high global productivity is anticipated, making the enzymatic process competitive with existing chemical synthesis.
31 citations
TL;DR: Aims: Optimization of Penicillin G acylase (PAC) production from a novel isolate of Bacillus sp.
Abstract: Aims: Optimization of Penicillin G acylase (PAC) production from a novel isolate of Bacillus sp.
Methods: Fermentation medium for PAC production was optimized using a two-level fractional factorial design with seven components.
Results: A maximum production of 9·5 U ml−1 of PAC was obtained in an optimized medium containing (g l−1): K2HPO4, 1·0; MgSO4·7H2O, 0·1; CaCl2·2H2O, 0·1; PAA, 2·0; tryptone, 5·0; yeast extract, 3·0; and sucrose, 50·0.
Significance and Impact of the Study: The two-step medium optimization resulted in a twofold increase in PAC production. Since the strain Bacillus sp. PGS10 produces a high level of PAC, it could be a potential candidate for industrial production of PAC.
31 citations
TL;DR: Periplasmic aggregation of the PGA precursor polypeptide limits PGA production by recombinant E. coli and this limitation can be overcome by addition in the medium of a non-metabolizable sugar, such as sucrose, or of glycerol.
Abstract: The Escherichia coli penicillin G amidase (PGA), which is a key enzyme in the production of penicillin G derivatives is generated from a precursor polypeptide by an unusual internal maturation process. We observed the accumulation of the PGA precursor polypeptide in the insoluble material recovered after sonication of recombinant E. coli JM109 cells grown at 26°C. The aggregated nature of the accumulated molecules was demonstrated using detergents and chaotrophic agents in solubilization assays. The periplasmic location of the aggregates was shown by trypsin-accessibility experiments performed on the spheroplast fraction. Finally, we showed that addition of sucrose or glycerol in the medium strongly reduces this periplasmic aggregation and as a consequence PGA production is substantially increased. Thus, periplasmic aggregation of the PGA precursor polypeptide limits PGA production by recombinant E. coli and this limitation can be overcome by addition in the medium of a non-metabolizable sugar, such as sucrose, or of glycerol.
31 citations
TL;DR: Analysis of the crystal structure of PA has shown that residues alphaR145 and alphaF146 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.
31 citations
TL;DR: Results on the E .
Abstract: Penicillin amidase (EC 3.5.1.1 1) is an enzyme that can catalyze the hydrolysis and synthesis of amide bonds between phenylacetic or phenoxyacetic acid and their derivatives and 6-aminopenicillanic acid (6-APA) or 7-aminocephalosporanic acid (7-ACA) and its derivatives.' Penicillin amidases with different molecular weights (60-260 kDa), substrate specificities and pH optima have been found in more than 100 microorganisms. The biological function of these enzymes is still not known. The enzymes from E . coli (penicillin G amidase) and P. ostreatus (penicillin V amidase) are currently used on an industrial scale for the hydrolysis of penicillin G and V2 respectively. The main product-6-APA-is used for the synthesis of semisynthetic penicillins and cephalosporins. These enzymes can also be used as biocatalysts for the synthesis of the semisynthetic 8-lactam antibiotics. Two different processes can be applied here.3 In the equilibrium controlled process the enzyme is used as a hydrolase in the reverse direction that accelerates the condensation reaction to its thermodynamic equilibrium. The product yield cannot be influenced by the properties of the enzyme. In the second process-kinetically controlled-activated substrates must be used. The enzyme is here used as a transferase that catalyzes the transfer of the side-chain to the P-lactam nucleophile (6-APA, 7-ACA etc.). The maximum product yield in this process is a function of the properties of the enzyme.' Thus, for the kinetically controlled process the enzyme properties that influence the yield must be known. To obtain this information studies using pure enzyme are required. Some results on the E . coli penicillin amidase pertinent in this context are presented here.
31 citations