Showing papers on "Penicillin amidase published in 1994"
TL;DR: Accumulation of PA precursor protein suggests that postranslational processing and translocation through the cytoplasmic membrane limit PA production.
53 citations
TL;DR: Abstracts of the XV Chilean Congress of Microbiology, Valdivia, Chile, 9- 12 October, 1992, p. 67: studies on microbial pe&illih amidaie (IV): the production of penicillin amidase from a partially constitutive mutant of
35 citations
TL;DR: This extraction procedure was optimized, and it was found that 95% of the enzyme was extracted after a 10 m/M EDTA plus 10 mM guanidine treatment at room temperature for 10 h, which could avoid purification steps for particular applications.
Abstract: Penicillin acylase is an enzyme that allows transformation of natural penicillins. It hydrolyzes amides of phenylacetic acid and some closely related aryl-aliphatic acids.7 Under appropriate conditions it can also catalyze the production of semisynthetic penicillins from 6-aminopenicillanic acid. Though penicillin acylase is widely distributed among bac- teria and fungi, recombinant strains have been engineered to achieve higher production levels, among them, the E. coli strain G27 1 developed by Genetica (Rhbne-Poulenc Santk, France) which was used in this study. Methods for recovering proteins usually involve cell dis- ruption by osmotic shock or sonication or enzymatic diges- tion of bacterial walls with lysozyme. This normally implies protein losses by degrading enzymes such as proteases and a number of further steps for protein purification or the use of expensive or energy-consuming procedures and conse- quently product price increases. According to a number of researchers,2.3.7,9.13.
32 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: It is demonstrated, in contrast with that proposed by other authors, that the generation of gradients of pH inside the porous structure of very active enzyme derivatives may be not a problem but a ‘very profitable tool’ to improve the whole set of industrial parameters.
Abstract: We have developed integrated studies of enzyme reaction engineering for the hydrolysis of penicillin G catalysed by very active penicillin G acylase (PGA) derivatives. We have studied the distinct effect of a key variable (pH) on different industrial parameters (e.g. activity/stability parameters). In this way we have demonstrated, in contrast with that proposed by other authors, that the generation of gradients of pH inside the porous structure of very active enzyme derivatives may be not a problem but a ‘very profitable tool’ to improve the whole set of industrial parameters. In this way we can establish two distinct ‘optimal pH values’: (i) the one inside the particle of the biocatalyst and (ii) the one in the bulk solution. The use of an external pH of 8.0 associated with the promotion of a controlled decrease in internal pH (e.g. around a mean value of 5.5) was very useful to simultaneously obtain interesting values of all industrial parameters: (i) very high hydrolytic yields (higher than 97%); (ii) a very important increase on the stability of PGA derivatives (higher than a 50-fold factor); and (iii) a very small decrease in operational activity (approximately 15%) as compared with the one of soluble enzyme at pH 8.0 with no diffusional hindrances.
30 citations
TL;DR: The nucleotide (nt) sequence of the gene encoding penicillin G amidase of Arthrobacter viscosus strain ATCC 15,294 was determined and shows significant homology with other so far identified beta-lactam amidases of Gram- bacteria.
28 citations
25 citations
TL;DR: In this paper, the authors investigated the factors that affect the conversion of 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) to cephalexin, such as pH, temperature, concentrations of 7ADCA, d (−)-phenylglycine methyl ester (PGME), and immobilized penicillin G acylase (IMPGA), time, and molar ratios.
25 citations
TL;DR: It was shown that protein suspension stabilization, precipitation, and enzyme denaturation were affected by both protein and polymer concentration and mostly depended on the particular polymer-enzyme system.
24 citations
TL;DR: Several monoclonal antibodies have been prepared and immobilized for the biospecific isolation of penicillin amidase (PA) from Escherichia coli and only one of these mABs was found to be suitable for preparative bioaffinity chromatography of PA within the pH stability range.
22 citations
TL;DR: In this paper, a method is described which allows to evaluate analyte concentrations from flow-injection analysis signals of a pH field effect transistor (pH-FET) detector independently of the buffer capacity of the probe.
TL;DR: Escherichia coli cells with penicillin acylase activity were permeabilized with aqueous solutions of the cationic detergent N-cetyl-N,N, N,N-trimethylammonium bromide, at pH 8.0 and the activity was found to have almost doubled.
Abstract: Escherichia coli cells with penicillin acylase activity were permeabilized with aqueous solutions of the cationic detergent N-cetyl-N,N,N-trimethylammonium bromide (CTAB), at pH 80 and the activity was found to have almost doubled The concentration of CTAB, the time and temperature of treatment were optimised for maximum enzyme activity and were found to be 02%, 20 min and 5°C respectively Subsequently, the cell bound activity was retained for a longer period by chemical cross-linking with 01% glutaraldehyde
TL;DR: The presented method for protein design of processed enzymes, like PGA, can be applied to combine enzyme properties from different species for special applications to create a hybrid enzyme which was catalytically active.
Abstract: A cross-species penicillin G amidase (PGA) gene (pac) coding for an α-peptide and a linker peptide fromK. citrophila ATCC 21285 and a β-peptide fromE. coli ATCC 11105 has been constructed and cloned inE. coli. The naturally occurring PGA specific processing pathway led to the formation of a hybrid enzyme which was catalytically active. In comparison with the two wild-type enzymes the hybrid PGA was found to have higherk
cat values for the three tested substrates benzylpenicillin, ampicillin and 6-nitro-3-phenylacetamido-benzoic acid (NIPAB).K
m was between the values of the wild-type enzymes or close to that ofK. citrophila. The presented method for protein design of processed enzymes, like PGA, can be applied to combine enzyme properties from different species for special applications.
TL;DR: Bacillus sphaericus (NCIM 2478) produced high levels of penicillin V acylase when grown on cornsteep liquor — minerals medium at 25°C for 20 h andSupplementation of the medium either with 1 % (w/v) whole wheat bran or its aqueous extract brought about more than 70 % increase in the total enzyme activity.
Abstract: Bacillus sphaericus (NCIM 2478) produced high levels of penicillin V acylase (100 U / g dry cells) when grown on cornsteep liquor — minerals medium at 25°C for 20 h. Supplementation of the medium either with 1 % (w/v) whole wheat bran or its aqueous extract brought about more than 70 % increase in the total enzyme activity. Moreover, deletion of Na2HPO4, MgSO4, CaCl2, FeSO4, CuSO4 and KCl from the medium affected neither growth nor enzyme production.
TL;DR: It is shown that when glutaraldehyde is used for immobilization of this enzyme, the yield of immobilization is low and the immobilized enzyme activity loss is less than 10%.
TL;DR: Taking into account the amount of PGA mRNA present in the cells at 37°C, one would expect the production of active PGA at this temperature, but this is not the case and expression is blocked at a step after transcription.
Abstract: Escherichia coli ATCC 11105 and JM109, transformed with a multicopy plasmid carrying the penicillin G amidase (PGA) gene, were grown at 26 degrees and 37 degrees C, in the presence or the absence of phenylacetic acid (PAA) or of glucose. A method based on primer extension was developed to quantify in vivo levels of PGA mRNAs. A unique transcription start site was found to be used in all the fermentation conditions tested. This site is located 28 nucleotides upstream of the initiation codon. Its utilization is subjected to catabolic repression and is induced by PAA. This site is used at 37 degrees C, but the PGA mRNA level in E. coli ATCC 11105 is lower at 37 degrees C than at 26 degrees C. Induction of the pga gene by PAA was found to be more efficient in the producer strain. Taking into account the amount of PGA mRNA present in the cells at 37 degrees C, one would expect the production of active PGA at this temperature. This is not the case. Thus, at 37 degrees C, expression is blocked at a step after transcription.
TL;DR: The force of interaction between the enzyme and the metal‐chelate sorbent was low, and Pen G competed for binding sites at high concentrations, resulting in low activities of the immobilized enzyme.
Abstract: Penicillin G amidohydrolase (PGA) was immobilized on Cu(II)-chelate regenerable sorbents. A long spacer was essential for binding, such as bisoxirane in the case of Sepharose 4B or glycidoxypropyltrimethoxysilane in the case of silica-based carriers. The stability of the PGA-carrier was determined both by the interaction forces between PGA and the metal-chelate sorbent and the presence of penicillin G (Pen G). The force of interaction between the enzyme and the metal-chelate sorbent was low, and Pen G competed for binding sites at high concentrations. The carrier with a small pore size demonstrated diffusion restrictions during immobilization of PGA, resulting in low activities of the immobilized enzyme. This carrier could not be completely regenerated. Carriers with an average pore size of 55 nm or larger displayed fewer diffusion restrictions. The corresponding Cu(II)-chelate sorbents were regenerated several times.
TL;DR: The experimental kinetic data were obtained by measurement of the thermometric signal in the microcalorimetric column with immobilized enzyme and described by the introduced mathematical model involving the mass transfer and reaction kinetic phenomena.
Abstract: Flow microcalorimeter was used for the study of microkinetic properties of Escherichia coli cells enriched with the penicillin G acylase activity immobilized in calcium pectate gel. The experimental kinetic data were obtained by measurement of the thermometric signal in the microcalorimetric column with immobilized enzyme and described by the introduced mathematical model involving the mass transfer and reaction kinetic phenomena.
TL;DR: The design and characterization of a biocatalyst with whole cells of E. coli containing penicillin acylase activity were carried out and the catalyst was characterized in terms of its kinetic and diffusional properties as a function of particle size, as well as its storage and operational stability.
TL;DR: In this paper, the substrate specificity of immobilized penicillin-G acylase towards cephalosporin derivatives was studied, where the phenylacetyl meiety can be altered at the @-position with several small substitutes.
TL;DR: In this paper, 2-benzoxazolon-3-yl-acetic acid polyethylene glycol conjugates of variable properties were synthesized by means of the dicyclohexyl carbodiimide procedure in tetrahydrofuran.
TL;DR: Immobilised Penicillin G acylase from E. coli hydrolyses penicillin and cephalosporin derivatives protected at the carboxy group as the phenylacetoxymethylene esters as the Phenoxyacetyl moiety.
Abstract: Immobilised Penicillin G acylase from E. coli hydrolyses penicillin and cephalosporin derivatives protected at the carboxy group as the phenylacetoxymethylene esters. The corresponding hydrolysis of penicillin V retains the phenoxyacetyl moiety. Kinetic data of the hydrolysis are reported.
Journal Article•
TL;DR: B bentonite I as an absorbent according to 0.6% (w/v) showed a high recovery yield of enzyme activity, and it can be directly used for isolation and purification of penicillin acylase from the fermentation broth.
TL;DR: In this paper, N 6 -Phenylacetyl-2′-deoxyadenosine and N 2 -phenyl-acetyl 2′deoxyguanosine are readily deprotected in reactions catalyzed by free and immobilized penicillin amidase at pH 7.8 and 25°C.
Abstract: N 6 -Phenylacetyl-2′-deoxyadenosine and N 2 -Phenylacetyl-2′-deoxyguanosine are readily deprotected in reactions catalyzed by free and immobilized penicillin amidase at pH 7.8 and 25°C.
TL;DR: A quick method to evaluate the “crude” substrate susceptibility of the acyl moieties, by carrying out the reaction between a nucleophilic nucleus (penicillinic or cephalosporanlc) and an activated derivative of aryl acetic acids, by means of hydrolysis rate measurements of the selected derivative.
Abstract: INTRODUCTION In the synthesis of p-lactam antibiotics, the application of enzymatic catalysts has become more and more relevant. The steps directly involved in the use of penicillin amidase (PA) in the synthetic pathway to these target molecules are those for the preparation of 6-APA and 7-ADCA, and more recently for the direct synthesis of the exe-cyclic amino bond ~~3.4.5, s). Encouraging results in the latter reaction were reached using penicillin amidases and amino acid hydrolases from various strains. The most frequently used micro-organism is E. co/i, either as a first source of PA or as a producer of amidases cloned from other strains. Penicillin amidases from E. co/i and the phylogenetically related enzymes showed a wide versatility when used as catalysts far from their natural functions. Those enzymes have a fairly constricted substrate susceptivity toward the acyl moiety. The kinetic and mechanistic consequences coming from the structural morphology of the involved molecules were the subject of several publications V. 8. IO. 11). The need to concentrate our efforts on non-sterile approaches to the synthesis of penicillins and cephalosporins led us to develop a quick method to evaluate the “crude” substrate susceptibility of the acyl moieties, by carrying out the reaction between a nucleophilic nucleus (penicillinic or cephalosporanlc) and an activated derivative of aryl acetic acids, by means of hydrolysis rate measurements of the selected derivative. The chosen condition had to allow an evaluation of the relative enzymatic activity expressed over a series of molecules. This parameter provides an indirect evaluation of the acyt moiety fit to the active site of the enzyme, but it does not give conclusive information about the distribution between the products of the reaction with either a nucleophilic group or water p, 31. 13). In experimental systems involving penicillinic or cephalosporanic nuclei, the values of hydrolytic activity allow a first screening of the chances of success in relation to sets of values of the fundamental parameters of the reaction (14, IS).