Showing papers on "Penicillin amidase published in 1991"
TL;DR: Clues to the biological role of this enzyme have been provided, as well as new strategies for the commercial production and utilization of PA, revealing a variety of interesting features that are unique among microorganisms.
106 citations
TL;DR: The optimal experimental conditions for antibiotic synthesis catalysed by PGA were established, as a compromise solution, in order to obtain good values for every parameter of industrial interest.
88 citations
TL;DR: In this article, the synthesis of cephalothin from thienylacetic acid (TAA) and 7-aminocephalosporanic acid (7ACA) was carried out in the presence of high concentrations of organic cosolvents (e.g., 50% N,N′dimethyl-formamide) and under a wide range of experimental conditions (pH, temperature, etc.) by using very active and highly stabilized derivatives of Penicillin G acylase.
Abstract: Synthesis of cephalothin from thienylacetic acid (TAA) and 7-aminocephalosporanic acid (7ACA) has been carried out in the presence of high concentrations of organic cosolvents (e.g., 50% N,N′dimethyl-formamide) and under a wide range of experimental conditions (pH, temperature, etc.) by using very active and highly stabilized derivatives of Penicillin G acylase. We have been able to find the compromising solutions under which: (a) synthetic yields were markedly increased compared to those obtained in fully aqueous medium, (b) derivatives preserved a good percentage of catalytic activity, (c) derivatives were quite stable, and (d) high concentrations of substrates could be used. Under optimal conditions, 50 mM solutions of 7ACA in the presence of a slight excess of TAA were converted to cephalothin with yields higher than 95% and final concentrations of product up to 20 g/L were obtained.
41 citations
TL;DR: The stabilizing effect of pen G SO allowed the preparation of derivatives of these PGAs preserving full catalytic activity in spite of being 1,400- and 650-fold more stable than the corresponding soluble or one-point attached immobilized enzymes.
30 citations
TL;DR: The whole cell immobilization technique has been optimized for different process parameters and the granular catalyst has good mechanical strength, low protein leachability, and high retention of penicillin amidase activity.
24 citations
TL;DR: Competition experiments support the proposal that the linker endopeptide in the precursor serves to increase the probability of productive collision between folded alpha- and beta-peptides, and accounts for the basis of the temperature regulation of PA production in vivo.
Abstract: The in vitro folding and assembly of penicillin acylase (EC 3.5.1.11) (PA) to active enzyme has been studied. PA is a large bacterial protein (Mr = 86,000) comprising two peptides, alpha and beta, produced by proteolytic processing and activation of a 92-kDa precursor. Proteins that result from proteolytic processing are characteristically difficult if not impossible to refold. Different factors that affect folding and assembly of PA, including pH, ionic strength, and temperature, have been studied. Yields of 60% can be obtained, based on recovery of enzyme activity, together with another 20% of folded and associated monomer with conformation closely similar to that of the active enzyme but with the active site not formed. Evidence is presented for in vitro assembly proceeding via initial folding of the N-terminal alpha-peptide with subsequent collapse of the transiently folded beta-chain on to the surface of the former. A slow process of rearrangement follows association in vitro. Competition experiments support the proposal that the linker endopeptide in the precursor serves to increase the probability of productive collision between folded alpha- and beta-peptides. The effect of raised temperature is to interfere with the folding of the alpha-peptide, thus preventing proper folding of the precursor. This finding accounts for the basis of the temperature regulation of PA production in vivo.
23 citations
TL;DR: The site of reaction of penicillin acylase from Kluyvera citrophila with the potent inhibitor phenylmethanesulphonyl fluoride was investigated by incubating the inactivated enzyme with thioacetic acid to convert the side chain of the putative active-site serine residue to that of cysteine.
Abstract: The site of reaction of penicillin acylase from Kluyvera citrophila with the potent inhibitor phenylmethanesulphonyl fluoride was investigated by incubating the inactivated enzyme with thioacetic acid to convert the side chain of the putative active-site serine residue to that of cysteine. The protein product contained one thiol group, which was reactive towards 2,2'-dipyridyl disulphide and iodoacetic acid. Carboxymethylcysteine was identified as the N-terminal residue of the beta-subunit of the carboxy[3H]methylthiol-protein. No significant changes in tertiary structure were detected in the modified penicillin acylase using near-u.v. c.d. spectroscopy. However, the catalytic activity (kcat) with either an anilide or an ester substrate was decreased in the thiol-protein by a factor of more than 10(4). A comparison of sequences of apparently related acylases shows no other extensive regions of conserved sequence containing an invariant serine residue. The side chain of this residue is proposed as a candidate nucleophile in the formation of an acyl-enzyme during catalysis.
20 citations
TL;DR: Penicillin G acylase gene from Bacillus megaterium ATCC 14945 has been isolated and purified enzyme from subcloned B. subtilis showed that the native enzyme consisted of two identical subunits, each with a molecular weight of 57,000.
17 citations
TL;DR: This immobilization method for penicillin acylase prepared by glutaraldehyde crosslinking on hydrogenated porous polyacrylonitrile fibers was superior to other methods in specific activity and immobilization yield.
15 citations
TL;DR: The absorption and expression of the penicillin G acylase was dependent on pore volume, and maximum expression of 57% of adsorbed enzyme was obtained on beads synthesized with 40 mL of kerosene, indicating minimum pore-diffusion limitations.
Abstract: Adsorption and expression of penicillin G acylase was studied on macroporous methacrylate polymer beads of differing pore volume, generated with kerosene. The absorption and expression of the penicillin G acylase was dependent on pore volume. Maximum expression of 57% of adsorbed enzyme was obtained on beads synthesized with 40 mL of kerosene, indicating minimum pore-diffusion limitations.
10 citations
TL;DR: In this investigation cell-free culture filtrate was used instead of process water and an extracellular protein factor was found to influence the synthesis of penicillin amidase.
TL;DR: To obtain a catalyst with good mechanical stress stability and other operational characteristics, cross-linked aggregates of whole Escherichia coli containing penicillin amidase were reinforced with surface modified precipitated silica and chitosan.
Abstract: To obtain a catalyst with good mechanical stress stability and other operational characteristics, cross-linked aggregates of whole Escherichia coli containing penicillin amidase were reinforced with surface modified precipitated silica and chitosan. The immobilized cells plus mixed fillers possess better performance characteristics i.e. higher stability at 4°C and 30°C, least protein leaching capacity and good settling characteristics. Most suitable conditions to prepare catalyst with mixed fillers were: chitosan, 0.3: silica, 0.2 g/g d.wt cell; and minimum moisture content in catalyst, 30% (w/w).
TL;DR: A crucial role of the hydrophobicity of alumina in adsorption and catalysis by, adsorbed penicillin G acylase was investigated on porous alumina and alumina treated with polyethyleneimine.
Abstract: Immobilization of penicillin G acylase was investigated on porous alumina and alumina treated with polyethyleneimine. The latter treatment increased the percent expression by 63 % and decreased the adsorption by 40 % indicating a crucial role of the hydrophobicity of alumina in adsorption and catalysis by, adsorbed penicillin G acylase.
Journal Article•
TL;DR: The sequence- and stereospecificity of the S1- and S' i-subsites of bovine alpha-chymotrypsin and trypsin, proteinase K and penicillin amidase from E. coli and A. viscosus has been determined by hydrolysis and kinetically controlled peptide synthesis using different substrates.
Abstract: The sequence- and stereospecificity of the S1- and S' i-subsites (i = 1-3) of bovine alpha-chymotrypsin and trypsin, proteinase K and penicillin amidase from E. coli and A. viscosus has been determined by hydrolysis and kinetically controlled peptide synthesis using different substrates. The data are compared with results for other serine proteases and the thiol protease papain. The stereospecificities differ by orders of magnitude, decreased when the enzyme was immobilized and were influenced when organic solvent molecules were bound to the enzyme.
TL;DR: Penicillin amidohydrolase productivity of four locally isolated bacterial species is described and highest enzyme productivity of S. lutea was recorded.
Abstract: Penicillin amidohydrolase productivity of four locally isolated bacterial species is described. Organisms were identified asEscherichia coli, Pseudomonas aeruginosa, Sarcina lutea andBacillus megaterium. Highest enzyme productivity of 3.2 U/mL with a corresponding dry cell mass of 4.5 g/L was recorded fromS. lutea.
01 Mar 1991
TL;DR: It is evident that the precursor processing step(s) is sensitive to temperature, and the transcription, translation, and post-translational processing of PGA gene expression is reported.
Abstract: The expression of penicillin G acylase (PGA) gene is sensitive to temperature. Cells of E. coli DH 5 alpha harboring a plasmid pWGA carrying a gene for PGA produce active PGA at low level at 37 degrees C, but can synthesize significant amount of the enzyme below 30 degrees C. Active PGA is formed from a 94 KD precursor processed through remove of a signal peptide and a spacer peptide to yield an enzyme that contain a 23 KD (alpha) and 65 KD (beta) subunit. This paper reported the effect of temperature on transcription, translation, and post-translational processing of PGA gene expression. We detected the transcription of PGA gene by Northern blot, and found the amount of PGA mRNA at 37 degrees C much more than that 30 degrees C when the cells of DH 5 alpha (pWGA) were cultured. It is evident that the precursor processing step(s) is sensitive to temperature.
01 Jan 1991