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PubMed2004

Molecularly imprinted solid phase extraction-pulsed elution-mass spectrometry for determination of cephalexin and alpha-aminocephalosporin antibiotics in human serum.

A highly selective molecularly imprinted solid phase extraction (MISPE)-pulsed elution (PE) method coupled with electrospray mass spectrometry (MS) was developed for the rapid screening and determination of cephalexin in alpha-aminocephalosporin antibiotics. This method involved the solid phase extraction of cephalexin using a molecularly imprinted polymer micro-column, and pulsed elution with 1% trifluoroacetic acid in methanol, which contains sulindac as an internal standard for enhanced precision in MS detection. An LC/MS spectrometer was operated in the positive electrospray mode, and the selected-ion-recording (SIR) function was employed to detect the molecular ions of cephalexin, cefradine, cefadroxil and sulindac at m/z 348, 350, 363 and 357. Linearity was achieved in the cephalexin concentration range from 0.3 to 25microg/ml (or 5-500ng) (R(2) = 0.998). The detection limit was estimated at 0.04microg/ml (or 0.8ng) of cephalexin. Advantages of the newly developed MISPE-PE-MS, over the previously reported MISPE-DPE-FPE-UV, were evidenced in terms of detection limit, analysis time, solvent consumption, and simplicity of method development.

Cephalexin↗
PubMed2004

Direct visualization of peptide uptake activity in the central nervous system of the rat.

Carrier-mediated transport of small peptides and peptidomimetics offers the opportunity for a targeted drug delivery across cell membranes in the central nervous system (CNS). This process is mediated by the proton-coupled transporter PEPT2 which is expressed in glial and choroid plexus cells. In the present studies, an uptake assay was established to visualize directly peptide uptake in intact rat brain slices. Accumulation of a reporter molecule, the fluorophore-labeled dipeptide derivative D-Ala-L-Lys-AMCA, was found in plexus choroideus and glial cells and uptake was inhibited by prototypical PEPT2 substrates, such as glycyl-L-glutamine and cefadroxil. The presence of PEPT2 was confirmed by RT-PCR and Northern blot analysis. This first CNS peptide and drug transport-visualizing assay may be used to examine new compounds which are carried by the proton-driven CNS peptide transporter.

Animals↗
PubMed1997

Structural specificity requirements in the binding of beta lactam antibiotics to human serum albumin.

The binding of some cephalosporins of pharmacological interest, to human serum albumin was studied using ultrafiltration method. The identification of the binding sites in albumin was also performed using probes for the so-called sites I, II, bilirubin and fatty acids binding sites. Cephalosporins were classified into three groups according to their affinity for albumin: low affinity (K = 10-10(2) M-1), medium affinity (K = 10(3) M-1) and high affinity (K = 10(4) M-1). Cephalosporin binding to albumin produced a perturbation of several basic amino acids of the protein such as histidine and lysine. It was found that only cefuroxime, ceftazidime and cefoperazone interact slightly with site I on serum albumin, while site II possesses capacity to bind: cephradine, cephalexin, ceftazidime, ceftriaxone, cefoperazone, cefaclor and cefsulodin. The bilirubin binding site showed capacity to interact with a great number of cephalosporins: ceftriaxone, cefazolin, cephaloglycin, cefamandole, cefotaxime, cefoxitin, cefuroxime, cefoperazone and cefadroxil. Ceftriaxone showed capacity to bind to the fatty acid binding site on HSA. No relation was found between the displacement of the marker and the chemical nature of the substituents at R1 and R2. Cephalosporins interact with HSA at the binding region that involves: tyrosyl 411, histidyl 146 and lysyls 195, 199, 225, 240 and 525 residues. The chemical modification of specific amino acids showed that the interaction of these amino acids with beta lactam antibiotics is not carried out to the same extent for all the cephalosporins tested. The results obtained revealed that the binding sites for cephalosporins on albumin are structurally heterogeneous, having different amino acids in the vicinity of the ligand molecule.

Acid-Base Equilibrium↗
PubMed1997

Community-acquired Serratia marcescens meningitis.

Serratia marcescens is an unusual cause of community-acquired meningitis in adults. We report a case of S. marcescens meningitis occurring 29 years after a head injury and preceded by 3 years of intermittent nasal discharge of cerebrospinal fluid (CSF). One month before admission, the patient had received treatment with cefadroxil. This case illustrates the risk of Gram-negative bacillary meningitis in patients with a CSF leak when they are treated with antibiotics. When patients have a chronic clear nasal discharge, one should look for a past medical history of head injury before prescribing antibiotics. In the presence of a fistula, any antibiotherapy may lead to the selection of resistant organisms which may be difficult to treat. Due to the high risk of meningitis and the fact that spontaneous closure in delayed CSF rhinorrhoea is unlikely, surgical repair of any associated fistulae is mandatory.

Anti-Bacterial Agents↗
PubMed1997

High-performance liquid chromatographic analysis of amoxicillin in human and chinchilla plasma, middle ear fluid and whole blood.

We extended the application of a sensitive high-performance liquid chromatography assay of amoxicillin developed in this laboratory for human plasma and middle ear fluid (MEF) to other sample matrices including chinchilla plasma or MEF and human and chinchilla whole blood with minor modification and validated the limit of quantitation at 0.25 microg/ml with a 50-microl sample size for human and chinchilla plasmas or MEFs. Amoxicillin and cefadroxil, the internal standard, were extracted from 50 microl of the samples with Bond Elut C18 cartridges. The extract was analyzed on a Keystone MOS Hypersil-1 (C8) column with UV detection at 210 nm. The mobile phase was 6% acetonitrile in 5 mM phosphate buffer, pH 6.5 and 5 mM tetrabutylammonium. The within-day coefficients of variation were 2.7-9.9 (n=4) and 1.7-7.2% (n=3) for chinchilla plasma and MEF samples, respectively; 2.8-8.1% (n=3) and 2.9-4.7% (n=3) for human and chinchilla whole blood, respectively. An alternative mobile phase composition for chinchilla plasma and MEF samples reduced the analysis time significantly.

Amoxicillin↗
PubMed2002

Selective spectrophotometric determination of phenolic beta-lactam antibiotics.

Two simple and selective spectrophotometric methods were developed for the quantitative determination of cefoperazone sodium, cefadroxil monohydrate, cefprozil anhydrous and amoxicillin trihydrate in pure forms as well as in their pharmaceutical formulations. The methods are based on the selective oxidation of these drugs with either Ce (IV) or Fe (III) in acid medium to give an intense yellow coloured product (lambda(max)=397 nm). The reaction conditions were studied and optimized. Beer's plots were obeyed in a general concentration range of 5-30 microg ml(-1) with correlation coefficients not less than 0.9979 for the four drugs with the two reagents. The methods are successfully applied to the analysis of pharmaceutical formulations containing amoxicillin, either alone or in combination with potassium clavulanate, flucloxacillin or dicloxacillin. They were also applied to the analysis of the other three studied drugs in vials, capsules, tablets and suspensions with good recovery; percent ranged from 99.7 (+/-0.46) to 100.32 (+/-1.05) in the Ce (IV) method and 99.6 (+/-0.50) to 100.3 (+/-1.32) in the Fe (III) method. Interferences from other antibiotics and additives products were investigated.

Anti-Bacterial Agents↗
PubMed1998

Cefuroxime selective electrodes for batch and FIA determinations in pharmaceutical preparations.

Different cefuroxime selective electrodes, without internal reference solution and comprising PVC membranes, were constructed and evaluated. Membranes were prepared with cefuroxime tetraoctylammonium (A) or cefuroxime bis(triphenylphosphoranylidene)ammonium (B) as ion-exchanger, 2-nitrophenyl octyl ether (X) or bis(2-ethylhexyl)sebacate (Y) as plasticizing mediator solvent and 4-tert-otcylphenol (TOP) as additive. From the comparative evaluation of the described electrodes, membranes comprising 2-nitrophenyl octyl ether, cefuroxime tetraoctylammonium and 4-tert-otcylphenol presented better working characteristics. For these electrodes (type XA-TOP), with a lifetime > 5 months, a lower limit of linear range of 2.8 x 10(-4) M, a practical detection limit of 1.3 x 10(-4) M, a reproducibility of approximately +/-0.6 mV day(-1) and a slope of -50.4 mV decade(-1), under H3PO4/NaH2PO4 solutions (pH 3.5; I =0.1 M), were found. The presence of the additive on the membranes was of crucial importance for the electrodes good characteristics. Interference from sulphate, chloride, nitrate, iodide, cefaclor, cefadroxil, cefazolin and cephradine, on the electrodes behaviour was evaluated. Only a slight interference from nitrate and iodide was recorded, being type XA-TOP electrodes the most selective units. Electrodes with a tubular configuration prepared with type XA-TOP membranes, aiming flow injection analysis, were also constructed. When these tubular potentiometric detectors were evaluated in a double-channel flow injection manifold, with 3.5 pH and 0.1 M ionic strength conditions, significantly better working characteristics than those of the corresponding conventional electrodes, namely higher slopes (-54.6 mV decade(-1)) and better reproducibilities (+/-0.2 mV day(-1)), were found. Both conventional and tubular type XA-TOP electrodes were used for injections analyses by batch and FIA, respectively, presenting low consumption of samples and reagents. Relative error deviations to the reference procedures <3.0% were found.

Cefuroxime↗
PubMed2003

Prevalence and antimicrobial susceptibility of pathogens in uncomplicated cystitis in Europe. The ECO.SENS study.

The ECO.SENS study investigated the prevalence and antimicrobial susceptibility of pathogens causing community-acquired acute uncomplicated urinary tract infections (UTIs) in 4734 women aged 18-65 years presenting with symptoms of acute UTI, at 252 community healthcare centres in 16 countries in Europe plus Canada. Resistance in Escherichia coli occurred most frequently to ampicillin (30%) and sulphonamides (29%), followed by trimethoprim (15%), trimethoprim/sulphamethoxazole (14%) and nalidixic acid (5%) but was low to co-amoxiclav, mecillinam, cefadroxil, nitrofurantoin, fosfomycin, gentamicin and ciprofloxacin, all at<3%. Consumption of antibiotics in 1997 varied more than 4-fold within Europe and from 9 to 37 DDD/1000 inhabitants/day, the consumption being highest in Southern Europe. The consumption of broad-spectrum penicillins correlated with resistance to ampicillin and there was a clear correlation between quinolone consumption and resistance to ciprofloxacin and nalidixic acid. The 4-fold difference in antibiotic consumption within Europe and the correlation to resistance emphasises the importance of controlling antibiotic usage.

Adolescent↗
PubMed1997

Penicillin-binding protein sensitive to cephalexin in sporulation of Bacillus cereus.

Cephalexin, cefaclor, cefadroxil, and cefotaxime strongly inhibited sporulation of Bacillus cereus ts-4 at 1 microgram/ml. Cephalexin was most inhibitory on the sporulation of B. cereus when the antibiotic was added at 3 h after induction of sporulation by nutrient downshift technique. Examination of 4',6-diamidino-2-phenylindole-stained cells by fluorescence-phase contrast microscopy showed that cephalexin inhibited the formation of asymmetric septum. By using [3H]penicillin, eight penicillin-binding proteins (PBPs) were detected from the cells of B. cereus ts-4. Among them, four PBPs were also detected in sporulating cells. Affinity of cephalexin to PBPs were measured indirectly by competition for subsequent binding of radioactive penicillin G. Cephalexin strongly bound to PBP 4 with molecular weight of 72,000 in sporulating cells.

Bacillus cereus↗
PubMed2005

Heterogeneity of the IgE response to allergenic determinants of cefaclor in serum samples from patients with cefaclor-induced anaphylaxis.

BACKGROUND: Beta-lactam antibiotics, such as cefaclor, may cause IgE-mediated anaphylactic reactions. However, the clinically available serologic test has not been widely accepted, and the antigenic determinants of these drugs are unclear. OBJECTIVE: To describe 4 cases of anaphylaxis caused by cefaclor in which a specific IgE response to cefaclor was demonstrated. METHODS: Four patients with anaphylaxis to cefaclor and 35 nonatopic controls never exposed to cefaclor were studied. Skin tests and oral challenges with this drug were performed. The specific IgE response to the antigenic determinant of cefaclor-human serum albumin (HSA) conjugate was compared in each patient. The serum specific IgE to cefaclor-HSA conjugate was detected using enzyme-linked immunosorbent assay (ELISA). Also, ELISA inhibition studies using various concentrations of cefaclor-HSA, HSA alone, and free cefaclor were performed, as were hapten inhibition studies using cefaclor, cephalexin, cefadroxil, ampicillin, ceftriaxone, and cefotaxime. RESULTS: Three patients showed high levels of serum specific IgE to cefaclor-HSA and marked inhibition patterns to free cefaclor and cefaclor-HSA conjugate on ELISA inhibition testing. Hapten inhibition testing in 3 individual serum samples showed 2 different patterns. In patient 3, significant dose-dependent inhibitions (up to 92%) were noted with additions of free cefaclor and cefaclor-HSA conjugate, and lesser inhibitions (up to 74%) were noted with cephalexin, which shares the aminobenzyl side chain. In patients 1 and 2, marked dose-dependent inhibitions were noted only with additions of cefaclor-HSA conjugate and free cefaclor, whereas minimal inhibitions were noted with the other 5 compounds. CONCLUSIONS: The specific IgE response to cefaclor-HSA conjugate in patients with cefaclor anaphylaxis occurs against the hapten, in which heterogeneity of the antigenic determinant was noted to depend on the individual.

Adolescent↗
PubMed1989

Transport characteristics of ceftibuten, a new oral cephem, in rat intestinal brush-border membrane vesicles: relationship to oligopeptide and amino beta-lactam transport.

Ceftibuten undergoes H+-coupled uphill transport across rat small intestinal brush-border membrane vesicles. The effects of amino acids, peptides, folate, and beta-lactams on the uptake of ceftibuten were examined. Uptake of ceftibuten was competitively inhibited by dipeptides or tripeptides. A counter-transport effect on ceftibuten uptake was observed in the vesicle preloaded with these peptides, and the transport was temporarily against a concentration gradient (overshooting). On the other hand, ceftibuten uptake was not changed by amino acids and a tetrapeptide. Therefore, ceftibuten is predominantly transported via the oligopeptide transport system in the brush-border membranes. The relationship of ceftibuten transport to folate and other oral antibiotics was also investigated. Cyclacillin, cephradine, and cefadroxil exhibited both inhibitory and countertransport effects, but folate, cefaclor, and cephalexin showed only a slight inhibitory effect. As the transport of cefaclor showed no uphill uptake in the presence of a H+ gradient and its H+ stimulated uptake was small, a H+ gradient-independent carrier-mediated system seems to participate in its transport. These findings suggest that two different carrier-mediated transport systems, H+ gradient dependent and independent, may exist for oral cephems.

Amino Acids↗
PubMed1999

Intestinal transport of beta-lactam antibiotics: analysis of the affinity at the H+/peptide symporter (PEPT1), the uptake into Caco-2 cell monolayers and the transepithelial flux.

PURPOSE: This study on the intestinal transport of beta-lactam antibiotics was undertaken to investigate the correlation between cellular transport parameters and the bioavailability. METHODS: Transport of 23 beta-lactam antibiotics was characterized by measuring their ability to inhibit the uptake of glycylsarcosine into Caco-2 cells, their uptake into the cells and their total flux across the cell monolayers. RESULTS: Ceftibuten and cyclacillin were recognized by PEPT1 with affinity constants comparable to those of natural dipeptides (K(i) = 0.3 and 0.5 mM, respectively). Cefadroxil, cefamandole, cephradine, cefaclor, cefuroxime-axetil, cefixime, cephalotin, cephalexin and ampicillin also interacted with PEPTI (K(i) = 7-14 mM). In contrast, cefapirin, cefodizime, cefuroxime, cefmetazole, ceftazidime, benzyl-penicillin, ceftriaxone, cefpirome, cefotaxime, cefepime, cephaloridine and cefsulodin displayed no affinity to the transport system (K(i) > 20 mM). The uptake into the cells and the transepithelial flux was highest for those beta-lactam antibiotics, which showed the strongest inhibition of [14C]Gly-Sar transport (p < 0.0001). Exceptions were cefuroximaxetil and cephalotin. CONCLUSIONS: The probability of oral bioavailability for beta-lactam antibiotics is mainly determined by their affinity to PEPTI. A threshold K(i) value of 14 mM with respect to Gly-Sar uptake is required.

Administration, Oral↗
PubMed1999

Changes in bacterial enzymes and PCR profiles of fecal bacteria from a patient with ulcerative colitis before and after antimicrobial treatments.

A 26-year-old female patient, suffering from recurrent attacks of ulcerative colitis accompanied by extraintestinal symptoms (erythema nodosum and pyoderma gangrenosum), was evaluated for the effect of antibacterial agents on the intestinal bacteria and their enzymatic activities. The enzymes were assayed both at the onset of disease symptoms and after treatment with each of five drug regimens (fluconazole and cefadroxil, cefuroxime axetil and cestriaxone sodium, ciprofloxacin and cestriaxone sodium, ciprofloxacin alone, and ciprofloxacin, metronidazole, and cephalexin). The activities of azoreductase, nitroreductase, oxidoreductase, glucuronidase, and sulfatase were generally lower following all of the treatments, especially when ciprofloxacin was included. The DNA from each sample was amplified by PCR, using random primers. Profiles of amplified DNA on agarose gels showed different patterns, indicating differences in the microflora before and after the antimicrobial treatments. The clinical response to antibacterial therapy was consistent with the decreased bacterial enzymatic activities and changes in the microbial population. Ciprofloxacin, which was associated with the most dramatic falls in enzymatic activity, also had the best clinical results. We conclude that intestinal bacteria and their enzymes play important roles in ulcerative colitis and that population changes can be monitored using PCR profiles.

Adult↗
PubMed1989

Contraluminal organic anion and cation transport in the proximal renal tubule: V. Interaction with sulfamoyl- and phenoxy diuretics, and with beta-lactam antibiotics.

In order to study the interaction of sulfamoyl- and phenoxy diuretics as well as of beta-lactam antibiotics with the contraluminal anion and cation transport systems the inhibitory potency of these substances against the influx of 3H-para-aminohippurate, 14C-succinate, 35S-sulfate and 3H-N1-methylnicotinamide into cortical tubular cells have been determined. 1.) 2-, 3- and 4-sulfamoylbenzoate inhibit contraluminal PAH influx. N-dipropyl substitution to yield probenecid or ring-substitution to yield furosemide and piretanide augment the inhibitory potency. However, hydrochlorothiazide and acetazolamide exert only a moderate inhibitory potency. Succinate transport was inhibited by furosemide only. Sulfate transport was inhibited by furosemide and 3-sulfamoyl-4-phenoxybenzoate as well as by probenecid, piretanide, hydrochlorothiazide and acetazolamide. 2.) Phenoxyacetate, -propionate, and -butyrate exert increasing inhibition against PAH transport. The weed-killers 2,4-dichloro-, and 2,4,5-trichlorophenoxyacetate (2,4 D and 2,4,5 T) had a similar inhibitory potency, while ethacrynic acid showed a lower and the uricosuric tienilic acid a higher inhibitory potency. None of the compounds of this group interact with contraluminal succinate transport, and only the multiring-substituted compounds 2,4 D, 2,4,5 T, ethacrynic and tienilic acid interact slightly with the sulfate transporter. 3.) The monocarboxylic penicillins benzylpenicillin and phenoxymethylpenicillin as well as the dicarboxylic ticarcillin interact with the contraluminal PAH transport. The aminopenicillin ampicillin had a lower, and apalcillin a higher inhibitory potency than monocarboxylic penicillin. Benzylpenicillin showed small inhibition against succinate transport and ticarcillin against sulfate transport. 4.) The monocarboxylic cephalosporine, 6315 S Shionogi, and the aminocephalosporines, cephalexin and cefadroxil, showed an app. Ki.PAH as the comparable penicillins. The zwitterions cephaloridine and cefpirome did not interact with the PAH transporter, but with the organic cation (NMN) transporter. Amongst the amino-thiazol-containing compounds cefotaxime, ceftriaxone, and cefodizime, increasing interaction with the PAH transporter was seen dependent of a second ionizable anionic group. Compounds with two ionizable anionic groups (cefsulodin, ceftriaxone, cefodizime) exert also a small inhibitory potency against sulfate transport. None of the cephalosporins interacted with the dicarboxylate transporter. The interaction pattern of the tested compounds is in accordance with the specificity requirements for the contraluminal transporters depending on electrical charge and hydrophobicity.

Animals↗
PubMed1990

Automated flow injection spectrophotometric determination of para- and meta-substituted phenols of pharmaceutical interest based on their oxidative condensation with 1-nitroso-2-naphthol.

The reaction of para- and meta-substituted phenols with 1-nitroso-2-naphthol in the presence of either CeIV or PbIV as an oxidant has been used to develop a fast automated flow injection (Fl) method. A stopped-flow kinetic study of the reaction revealed the optimum conditions for the proposed Fl method. Acetaminophen, amoxicillin, cefadroxil, isoxsuprine, nylidrin, propylparaben, tyrosine and metaraminol can be determined in the range 1 x 10(-4)-8 x 10(-4) M, with relative standard deviations of less than 2%, and a measurement throughput of 40 measurements h-1. The method was evaluated by performing interference studies of common excipients and assaying commercial formulations of acetaminophen and isoxsuprine. The results were in good agreement with those obtained by acceptable spectrophotometric or high-performance liquid chromatographic methods (mean difference 2.1%). The high sample throughput of the Fl method was exploited by performing a content uniformity test of isoxsuprine tablets.

Acetaminophen↗
PubMed1990

Continuous flow molecular emission cavity analysis of cephalosporins by alkaline degradation to sulphide.

A continuous flow method for the determination of some cephalosporins (cephradine, cephalexin, cephalosporin C, cefadroxil, cephapirin and cephalothin) in the general range 10.0-250.0 micrograms ml-1 is described. The sample is mixed with sodium hydroxide and remains for 20 min at 90 degrees C in the delay coil of an air-segmented system. The solution is then mixed with an excess of orthophosphoric acid, and the hydrogen sulphide evolved is continuously transferred into the cavity for generation of the S2 molecular emission. The analysis is automated, requires no sample pre-treatment and samples can be analysed at a rate of 30 per hour with a relative error of 1-2%. The method was evaluated by carrying out recovery experiments and by the analysis of commercial formulations. Results agreed well with those obtained by the standard methods.

Alkalies↗
PubMed2006

Positive effect of natural and negatively charged cyclodextrins on the stabilization of penicillins towards beta-lactamase degradation due to inclusion and external guest-host association. An NMR and MS study.

The complexation of penicillin (1a-c) and cephalosporin (2a,b) antibiotics with cyclodextrins (CDs), both natural [beta-CD (3b) and gamma-CD (3c)] and carboxylated [heptakis(6-oxycarbonylethylthio-6-deoxy)-beta-CD sodium salt (4b) and octakis(6-oxycarbonylethylthio-6-deoxy)-gamma-CD (4c) sodium salt], has been studied at neutral pH. Penicillins [ampicillin (1a), amoxicillin (1b) and dicloxacillin (1c) form inclusion complexes with the above CDs, as was shown by extensive NMR spectroscopic studies, whereas cephalosporins (cephalexin, cefadroxil) do not. Inclusion of the penicillins into either 3b or 4b was not accompanied by significant chemical shift changes in the 1H NMR spectra. On the contrary, with the wider 3c and its derivative 4c inclusion was evidenced by both chemical shift displacements of the cavity protons and intermolecular interactions, indicating the formation of primarily 1:1 guest-host inclusion complexes. The binding constants for 1a/3c, 1a/4c and 1c/3c were calculated as 19 +/- 4, 17 +/- 0.9 and 622 +/- 200 M(-1), respectively. With 4c, a 1:2 stoichiometry was also found. In addition, simultaneous formation of aggregates by external association takes place in solution, as shown by the ESI-mass spectrometric data. Studies on the hydrolysis of ampicillin under pseudo-first order conditions using an excess of 3c, 4c and of linear maltoheptaose at pH 7 showed that the drug hydrolysed at a similar rate in all cases. In the presence, however, of beta-lactamase enzyme and the carboxylated host 4c, ampicillin degraded twice as slowly (0.008 h(-1)) as in the presence of beta-lactamase alone (0.017 h(-1)). This was explained by the effective protection provided by both inclusion and external association of the host. The interaction, therefore, of penicillins with carboxylated CDs may present a means to lessen the chemical instability of these drugs in the presence of beta-lactamase enzymes.

Carbohydrate Conformation↗
PubMed2002

The role of hydrophobic active-site residues in substrate specificity and acyl transfer activity of penicillin acylase.

Penicillin acylase of Escherichia coli catalyses the hydrolysis and synthesis of beta-lactam antibiotics. To study the role of hydrophobic residues in these reactions, we have mutated three active-site phenylalanines. Mutation of alphaF146, betaF24 and betaF57 to Tyr, Trp, Ala or Leu yielded mutants that were still capable of hydrolysing the chromogenic substrate 2-nitro-5-[(phenylacetyl)amino]-benzoic acid. Mutations on positions alphaF146 and betaF24 influenced both the hydrolytic and acyl transfer activity. This caused changes in the transferase/hydrolase ratios, ranging from a 40-fold decrease for alphaF146Y and alphaF146W to a threefold increase for alphaF146L and betaF24A, using 6-aminopenicillanic acid as the nucleophile. Further analysis of the betaF24A mutant showed that it had specificity constants (kcat/Km) for p-hydroxyphenylglycine methyl ester and phenylglycine methyl ester that were similar to the wild-type values, whereas the specificity constants for p-hydroxyphenylglycine amide and phenylglycine amide had decreased 10-fold, due to a decreased kcat value. A low amidase activity was also observed for the semisynthetic penicillins amoxicillin and ampicillin and the cephalosporins cefadroxil and cephalexin, for which the kcat values were fivefold to 10-fold lower than the wild-type values. The reduced specificity for the product and the high initial transferase/hydrolase ratio of betaF24A resulted in high yields in acyl transfer reactions.

Aminobenzoates↗