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Showing papers on "Penicillin amidase published in 2005"


Journal ArticleDOI
TL;DR: Thermal inactivation studies showed that these multipoint enzyme-support attachments promoted an increase in the stability of the immobilized enzymes.
Abstract: The controlled and partial modification of epoxy groups of Eupergit C and EP-Sepabeads with sodium sulfide has permitted the preparation of thiol-epoxy supports. Their use allowed not only the specific immobilization of enzymes through their thiol groups via thiol–disulfide interchange, but also enzyme stabilization via multipoint covalent attachment. Penicillin G acylase (PGA) from Escherichia coli and lipase from Rhizomucor miehei were used as model enzymes. Both enzymes lacked exposed cysteine residues, but were introduced via chemical modification under very mild conditions. In the first moments of the immobilization, a certain percentage of immobilized protein could be released from the support by incubation with DTT; this confirms that the first step was via a thiol–disulfide interchange. Moreover, the promotion of some further epoxy-enzyme bonds was confirmed because no enzyme release was detected after some immobilization time by incubation with DTT. In the case of the heterodimeric PGA, it was possible to demonstrate the formation of at least one epoxy bond per enzyme subunit by analyzing with SDS-PAGE the supernatants obtained after boiling the enzyme derivatives in the presence of mercaptoethanol and SDS. Thermal inactivation studies showed that these multipoint enzyme-support attachments promoted an increase in the stability of the immobilized enzymes. In both cases, the stabilization factor was around 12–15-fold comparing optimal derivatives with their just-thiol immobilized counterparts. © 2005 Wiley Periodicals, Inc.

97 citations


Journal ArticleDOI
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


Journal ArticleDOI
TL;DR: It is shown that the pro-sequence in cis functions as a folding catalyst and accelerates the folding rate by seven orders of magnitude and that Ca2+, found in the crystal structure, is not directly involved in the folding process.

30 citations


Journal ArticleDOI
TL;DR: Compared to the penicillin acylase of E. coli, PAS2 showed superior potential for the synthesis of 6-aminopenicillanic acid (6-APA)-derived antibiotics, allowing the accumulation of up to 2.3-fold more target product at significantly higher conversion rates.

30 citations


Journal ArticleDOI
TL;DR: This modification improved the immobilization efficiency of PGA on glyoxyl agarose and the catalytic properties of the PGA derivative, although it impaired the posttranslational steps of overexpressed protein maturation.
Abstract: A tag of three lysines alternating with three glycines was added to the C-terminal end of the β chain of penicillin G acylase (PGA). This modification improved the immobilization efficiency of PGA on glyoxyl agarose and the catalytic properties of the PGA derivative, although it impaired the posttranslational steps of overexpressed protein maturation.

26 citations


Journal ArticleDOI
TL;DR: Polyethylene glycol (PEG 600)-ammonium sulphate aqueous two-phase systems have been studied with the aim to have, during the reaction, a continuous extraction of the acylation product outside of the enzyme environment (the ammonium sulphates phase).

26 citations


Journal ArticleDOI
TL;DR: The enzymatic synthesis of ampicillin from d -phenylglycine methyl ester and 6-aminopenicillic acid, using PGA from Escherichia coli, indicates that the acylation step may occur with 6-APA already positioned for the nucleophilic attack.
Abstract: Penicillin G acylase (PGA) catalyzes the synthesis/hydrolysis of acyl derivatives of phenylacetic acid through the formation of a covalent intermediate (the acyl–enzyme complex). When used for the kinetically controlled synthesis of β-lactam antibiotics, this enzyme promotes two undesired side reactions: the hydrolysis of the acyl side-chain precursor and of the antibiotic. Therefore, a high selectivity (synthesis/hydrolysis, S/H ratio) is very important for the process economics. Here, the enzymatic synthesis of ampicillin from d -phenylglycine methyl ester (PGME) and 6-aminopenicillic acid (6-APA), using PGA from Escherichia coli (EC 3.5.1.11) is studied. Kinetic assays provided S/H for high concentrations of substrates (up to 200 mM of 6-APA and 500 mM of PGME), using soluble PGA, at 25 °C, pH 6.5. S/H increased with 6-APA concentration, in accordance with the literature. However, when the concentration of 6-APA approached saturation, the rate of enzymatic hydrolysis tended towards zero (i.e., S/H tended to infinity). On the other hand, when the concentration of ester was augmented, S/H consistently decreased. This behavior, to the best of our knowledge still not reported, indicates that the acylation step may occur with 6-APA already positioned for the nucleophilic attack.

25 citations


Journal ArticleDOI
TL;DR: The objective of this work was to study the immobilization of penicillin G acylase from Escherichia coli on to chitosan–glutaraldehyde beads by multipoint covalent binding using a 23 experimental design.
Abstract: The objective of this work was to study the immobilization of penicillin G acylase from Escherichia coli on to chitosan-glutaraldehyde beads by multipoint covalent binding. This process was optimized using a 2(3) experimental design. The parameters selected for the present study were the concentrations of glutaraldehyde, phenylacetic acid and sodium borohydride. Three responses were chosen, namely immobilization yield and stabilization factors of enzyme derivatives at high temperature and at alkaline pH. All the runs at the maximum (+1) and minimum (-1) levels were performed at random. Three experiments were performed at the centre point, coded as zero, for experimental-error estimation. With respect to immobilization yield, the main effectors were the concentrations of glutaraldehyde and phenylacetic acid. For stabilization factors at 50 degrees C and at alkaline pH, the main effectors were the concentrations of glutaraldehyde and sodium borohydride and the interaction between them.

24 citations


Journal ArticleDOI
TL;DR: An unannotated protein from Bacillus subtilis has been expressed in Escherichia coli, purified and confirmed to possess penicillin V acylase activity, and the structure has been solved using molecular-replacement methods with B. sphaericus peniillin VAcylase (PDB code 2pva) as the search model.
Abstract: Penicillin acylase proteins are amidohydrolase enzymes that cleave penicillins at the amide bond connecting the side chain to their β-lactam nucleus. An unannotated protein from Bacillus subtilis has been expressed in Escherichia coli, purified and confirmed to possess penicillin V acylase activity. The protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 4 M sodium formate in 100 mM Tris–HCl buffer pH 8.2. Diffraction data were collected under cryogenic conditions to a spacing of 2.5 A. The crystals belonged to the orthorhombic space group C2221, with unit-cell parameters a = 111.0, b = 308.0, c = 56.0 A. The estimated Matthews coefficient was 3.23 A3 Da−1, corresponding to 62% solvent content. The structure has been solved using molecular-replacement methods with B. sphaericus penicillin V acylase (PDB code 2pva) as the search model.

20 citations


Journal ArticleDOI
TL;DR: Azlactone-functional dispersion and reverse phase suspension polymer supports were examined as immobilizing media for Penicillin G Acylase (PGA) in this paper, and the most effective supports were those that also contained primary and/or secondary amide functional groups.
Abstract: Azlactone-functional dispersion and reverse phase suspension polymer supports were examined as immobilizing media for Penicillin G Acylase (PGA). Results indicated that the most effective supports were those that also contained primary and/or secondary amide functional groups. A combination of hydrophobic interactions and hydrogen bonding between amide groups on the support and enzyme was proposed to provide important and intimate association between PGA and support prior to covalent coupling. Relative to an oxirane-functional commercial standard, the azlactone biocatalyst supports featured shorter coupling times and higher catalytic activities.

19 citations


Journal ArticleDOI
TL;DR: The culture medium for Streptomyces lavendulae ATCC 13664 was optimized on a shake-flask scale by using a statistical factorial design for enhanced production of penicillin acylalse and showed catabolite repression by different carbon sources such as glucose, lactose, citrate, glycerol, and glycine.
Abstract: The culture medium for Streptomyces lavendulae ATCC 13664 was optimized on a shake-flask scale by using a statistical factorial design for enhanced production of penicillin acylalse. This extracellularenzyme recently has been reported to bea penicillin Kacylase, presenting also high hydrolytic activity against penicillin V and other natural aliphatic penicillins such as penicillin K, penicillin F, and penicillin dihydroF,. The factorial design indicated that the main factors that positively affect penicillin acylase production by S. lavendulae were the concentration of yeast extract and the presence of oligoelements in the fermentation medium, whereas the presence of olive oil in the medium had no effect on enzyme production. An initial concentration of 2.5% (w/v) yeast extract and 3 μg/mL of CuSO4·5H2O was found to be best for acylase production. In such optimized culture medium, fermentation, of the microorganism yielded 289 IU/L of enzyme in 72 h when employing a volume medium/volume flask ratio of 0.4 and a 300-rpm shaking speed. The presence of copper, alone and in combination with other metals, stimulated biomass as well as penicillin acylase production. The time course of penicillin acylase production was also studied in the optimized medium and conditions. Enzyme production showed catabolite repression by different carbon sources such as glucose, lactose, citrate, glycerol, and glycine.

Journal ArticleDOI
TL;DR: Immobilization of penicillin acylase onto the capsule membranes resulted in increased operational stability of the enzyme and a very high enzyme activity, and the importance of capsular perstraction and reactive capsularPerstraction has been clearly demonstrated.
Abstract: The activity of penicillin acylase has been studied in aq. and org. solvents, as free enzyme as well as immobilized within the membrane of liq.-core capsules. The activity of the enzyme is inhibited by the accumulation of the products of the hydrolysis reaction, namely Ph acetic acid (PAA). In order to overcome this inhibition a range of org. solvents were tested for use in in situ product recovery. Of these solvents di-Bu sebacate (DBS) was chosen due to the rapid extn. rate, the high logP and to facilitate capsule prodn. The extn. efficiency at pH 3.5 for PAA was >80% for phase ratios of >50% free solvent with partition coeffs. of 8 and 0.7 for PAA and penicillin G (PenG), resp., thereby showing that PAA could be selectively extd. at pH 3.5 and 25 DegC. Liq.-core capsules contg. DBS were shown to efficiently remove PAA selectively and the PAA could be effectively backextd. and the capsules re-used in a three-stage process resulting in high product sepn. Immobilization of penicillin acylase onto the capsule membranes resulted in increased operational stability of the enzyme and a very high enzyme activity. Over 53.3% of the PAA formed could be recovered in the capsule core with a concn. over sevenfold higher than in the aq. phase. Higher extn. efficiencies could be obtained by varying the substrate concn. and no. of capsules. The enzyme immobilized on capsules could be stored for over 4 mo at pH 8 and 4 DegC with no loss of activity. Over 80% of the initial activity could be recovered over five repeated batch cycles of the bioconversion process. The importance of capsular perstraction and reactive capsular perstraction has been clearly demonstrated. [on SciFinder (R)]

Journal ArticleDOI
TL;DR: The non‐proportional correlation between increased mRNA longevity and amount of active enzyme propose that the rational strategies for yield improvement must be based on a simultaneous tuning of more than one yield restricting factor.

Journal ArticleDOI
TL;DR: In this work, immobilized PGA was utilized to catalyze the conversion of Ceph-G to 7-ADCA, and both inhibition kinetic mechanisms were different in that PAA showed noncompetitive inhibition to PGA whereas 7- ADCA appeared to be of competitive inhibition.

Journal ArticleDOI
TL;DR: The crystallization of three catalytically inactive mutants of penicillin V acylase (PVA) from Bacillus sphaericus in precursor and processed forms is reported, which will provide three-dimensional structures of precursor PVA forms, plus open a route to the study of enzyme-substrate complexes for this industrially important enzyme.
Abstract: The crystallization of three catalytically inactive mutants of penicillin V acylase (PVA) from Bacillus sphaericus in precursor and processed forms is reported. The mutant proteins crystallize in different primitive monoclinic space groups that are distinct from the crystal forms for the native enzyme. Directed mutants and clone constructs were designed to study the post-translational autoproteolytic processing of PVA. The catalytically inactive mutants will provide three-dimensional structures of precursor PVA forms, plus open a route to the study of enzyme–substrate complexes for this industrially important enzyme.

Journal ArticleDOI
TL;DR: In this paper, an equation for batch reactor performance was derived considering the kinetic mechanism proposed for the kinetically controlled synthesis of β-lactam antibiotics with penicillin acylase is favored at low temperatures and high cosolvent concentrations.

Journal ArticleDOI
TL;DR: High transcriptional activity of the natural promoter together with highpga gene dosage could result in a deleterious metabolic burden of the periplasmic enzyme.
Abstract: Recombinant plasmid pKA18 of the high expression bacterial system for penicillin amidase (‘penicillin G acylase’) bears the 3′ end region of IS2 element. The IS2 sequence replaces the −35 region of promoter ofpga and extends up to TAGTAT box at position −10 of the promoter region. It therefore forms a hybrid promoter ofpga ppgaHT. A natural promoterppgaWT was not detected on any recombinant plasmid isolated from recombinant strains ofEscherichia coli constitutively producing penicillin amidase. PCR fragments carrying both types of promoters were cloned into the promoter-probe vector pET2 to compare their transcriptional activity: the activity ofppgaWT was 5× higher than that ofppgaHT. The same nucleotide “G” localized 28 nucleotides upstream of the translation start point was identified as the respective transcription start point of both mRNAs. An attempt was made to place thepga gene cloned on a plasmid under the control of the natural promoter: not a single clone expressing penicillin amidase was found among 150 transformants. High transcriptional activity of the natural promoter together with highpga gene dosage could result in a deleterious metabolic burden of the periplasmic enzyme.

Journal ArticleDOI
TL;DR: A family of an enzymatically catalyzed reaction network was studied, which involves the hydrolysis of penicillin G by peniculin G acylase in an isothermal continuous flow stirred tank reactor (CFSTR), and the capacity of steady state multiplicity was extended to its family of reaction networks.
Abstract: A family of an enzymatically catalyzed reaction network was studied, which involves the hydrolysis of penicillin G by penicillin G acylase in an isothermal continuous flow stirred tank reactor (CFSTR). This system consisted of 10 coupled non-linear equations and was found to be capable of exhibiting computational multiple steady states. A set of kinetic parameters determined from the existing experimental data were used to compute a set of rate constants and two corresponding steady states. This suggested that multiple steady states may occur in the system studied. The phenomena of bistability, hysteresis and bifurcation were discussed. Moreover, the capacity of steady state multiplicity was extended to its family of reaction networks.