Acylase Activity Research Articles

Diminished production of prostaglandin E2 by monocytes of newborns is due to altered fatty acid membrane content and reduced cyclooxygenase activity.

To determine the biochemical basis for reduced PGE2 expression by cord blood monocytes relative to adult peripheral blood monocytes, fatty acid composition of cell membranes together with the amount and activity of enzymes involved in the cyclooxygenase pathway were investigated. Gas chromatographic analysis of mononuclear cell fatty acids showed a significant reduction in long chain fatty acids, including arachidonic acid. Acylase and phospholipase A2 activity, together with lipocortin 1 levels, were similar in cord and adult monocytes. No difference in amounts of the 70-kDa cyclooxygenase type 1 enzyme was observed between the monocyte sources. The 74k-Da cyclooxygenase type 2 enzyme was synthesized de novo upon LPS stimulation in comparable amounts in both cord and adult monocytes, with both monocyte types responding with equal sensitivity to LPS. The ability of microsomes, prepared from cord monocytes, to convert arachidonic acid into eicosanoids of the cyclooxygenase pathway was reduced. In contrast, the ability of acetylated microsomes to convert PGG2 to PGE2 was comparable, suggesting that the deficiency in PGE2 production by cord monocytes is due to reduced activity, specifically at the cyclooxygenase-active site rather than the peroxidase active site. In conclusion, it appears that both reduced fatty acid content and diminished cyclooxygenase activity are responsible for diminished eicosanoid production by cord monocytes.

Oxidative modification of a cephalosporin C acylase from Pseudomonas strain N176 and site-directed mutagenesis of the gene

A cephalosporanic acid acylase from Pseudomonas strain N176 catalyzes hydrolysis of both glutarylcephalosporanic acid and cephalosporin C to 7-amino-cephalosporanic acid. Chemical modification of the enzyme with acidic hydrogen peroxide was performed to investigate residues which play important roles in enzymatic activity. The activity of the enzyme was reduced to 76% of the original by oxidation. From protein chemical analysis combined with site-directed point mutagenesis, modification of Met-164 was found to correspond to the reduction in activity. To study the effect of Met-164 on the enzymatic character, we prepared mutant acylases in which Met-164 was replaced with several other amino acids and obtained the following data: (i) there existed a trend of mutation to noncharged hydrophilic residues, resulting in an increase of activity against glutarylcephalosporanic acid; (ii) the mutation of Met-164 to Gly and Ala resulted in the lowering of both Km values and the optimal pHs against glutarylcephalosporanic acid; (iii) the mutation to Leu enhanced cephalosporin C acylase activity; and (iv) the mutation to Gln improved the k(cat) value for glutarylcephalosporanic acid. In particular, the mutation to Gln resulted in a high rate of conversion of glutarylcephalosporanic acid to 7-amino-cephalosporanic acid under conditions similar to those of a bioreactor system. These results may indicate that Met-164 is located in or near the cephalosporin compound binding pocket on the enzyme.

A postfermentative stage improves penicillin acylase production by a recombinant E. coli

Active penicillin acylase is formed only after an in vivo post-translational processing of the polypeptide precursor. Such a maturation process is rare in procaryotes. In this work, incubation under aerated conditions, of whole recombinant E. coli cells after glucose depletion and growth cessation, i.e., during the postfermentative stage, consistently resulted in 2- to 4-fold increases in penicillin acylase activity. Such results suggest that penicillin acylase maturation occurs to a high extent even during the postfermentative stage. Accordingly, the effect of different incubation conditions, during the postfermentative stage, on penicillin acylase was determined. Incubation under anaerobic conditions resulted only in a 1.27-fold increase of enzyme activity, with respect to the end of the batch culture, whereas a 3- and 4- fold increase occurred during incubation under dissolved oxygen concentrations of 100 and 43% (with respect to air sat.), respectively. Only a small negative effect, on the maturation process, was observed during incubation with acetate concentrations above 0.6 g/L. No effect of pH, in the range of 6.0 to 8.0, was observed.

Screening and characterization of glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas sp.

Three screening methods were used to isolate GL-7-ACA acylase-producing strains. Three positive isolates were identified with Pseudomonas nitroreducens CCRC 11041 possessing the highest activity, against GL-7-ACA and GL-7-ADCA. No activity was detected when Ceph C or succinyl-7-ACA was used as substrate; glutaric acid was found to be inhibitory. CCRC 11041 could produce maximal GL-7-ACA acylase activity when cultivated on meat extract medium II. The enzyme had a pH optimum of 5.0 and a temperature optimum of 42°C.

Mutant Escherichia coli penicillin acylase with enhanced stability at alkaline pH

Increased stability at alkaline pH should be a valuable attribute for the utilization of penicillin acylase in bioreactors employed to convert penicillins into 6-aminopenicillanic acid, a precursor of semisynthetic penicillins. In these systems, base is added for pH control, which results in local alkaline conditions that promote enzyme inactivation. Hydrolysis and synthesis reactions are also pH dependent. Here, we report work in which the gene coding for Escherichia coli penicillin acylase was subjected to oligonucleotide-directed random mutagenesis at regions coding for amino acids predicted to be at the surface of the enzyme. The resulting mutant library, cloned in E. coli, was screened by a filter paper assay of the colonies for the presence of penicillin acylase activity with enhanced stability at alkaline pH. Characterization of one of the selected clones revealed the presence of a mutation, Trp431-Arg, which would presumably alter the surface charge of the protein. In vitro experiments demonstrated a near twofold increase in the half-life of the mutant enzyme when stored at pH 8.5 as compared with the wild-type enzyme, with a comparable specific activity at several pH values. In general, the mutant displayed increased stability toward the basic side in the pH-stability profile. (c) 1995 John Wiley & Sons, Inc.

Assay of penicillin acylase in organic media

The synthetic substrate 6-nitro-3-(phenylacetamido) benzoic acid (NIPAB) is an appropriate substrate for assaying penicillin acylase activity in reversed micellar systems of Aerosol - OT in isooctane. Accumulation of 6-nitro-3-aminobenzoic acid (NABA) produced by the enzymatic hydrolysis of NIPAB, followed by the increase in absorbance at 405 nm, was linear at 4 to 20 mM for up to 30 minutes and 15 °C to 40 °C.

Screening and characterization of microorganisms with glutaryl-7 ADCA acylase activity

A screening of microorganisms producing glutaryl-7ADCA acylase, an enzyme able to hydrolyse glutaric acid selectively from glutaryl-3-deacetoxy-7-aminocephalosporanic acid (glutaryl-7ADCA), has been carried out in soil samples. Five microorganisms expressing acylase activity were isolated and classified as Bacillus cereus, Achromobacter xylosooxidans, Bacillus sp., Pseudomonas sp. and Pseudomonas paucimobilis. The screening was carried out by preparing enrichment cultures containing glutaryl-7-ADCA or cephalosporin C as the selective carbon source. Four model compounds (adipoyl-, glutamyl- and glutaryl-p-nitroanilide and glutarylcoumarin), mimicking the glutaryl-7ADCA beta-lactam moiety, were synthesized as substrates suitable for the rapid screening of the microorganisms (2500) isolated from the enrichment cultures. A total of 300 strains were active on the model substrates and only 5 displayed acylase activity on glutaryl-7ADCA. The fermentation parameters, such as pH and inducer concentration, for the optimal acylase expression and acylase specificity towards the model substrates were different for each strain.

Screening and characterization of microorganisms with glutaryl-7ADCA acylase activity

Screening and characterization of microorganisms with glutaryl-7ADCA acylase activity

Recombinant whole cell penicillin acylase biocatalyst: Production, characterization and use in the synthesis and hydrolysis of antibiotics

The production of a whole cell biocatalyst with high specific penicillin acylase activity using a recombinant strain is described. High activities were obtained using exponentially fed-batch fermentations to overproduce the enzyme. The cells were then entrapped in agar and the activity characterized in terms of particle size and stability. The technical feasibility of the catalyst in synthesis and hydrolysis reactions was demonstrated.

In vitro antitumor activity of polymyxin acylase from Pseudomonas sp. M-6-3.

Polymyxin acylase from Pseudomonas sp. M-6-3 can deacylate not only polymyxin group antibiotics, but also the long-chain fatty acyl group of proteins and peptides. We found the in vitro antitumor activity of polymyxin acylase against murine and human tumor cells, especially KB cells. The mechanism of the antitumor activity remains equivocal, but we speculate that it may result from the affinity of polymyxin acylase for long-chain fatty acyl proteins in human carcinoma cells.

Enzymatic transformation of cephalosporin C to 7-amino-cephalosporanic acid. Part II: Single-step procedure using a coimmobilized enzyme system

The enzymatic transformation of cephalosporin C to 7-amino-cephalosporanic acid (7-ACA) using coimmobilized d-aminoacid oxidase (DAAO) and 7-β-(4-carboxybutanamido)cephalosporanic acid acylase (Gl-7-ACA acylase) is reported. The results from the coimmobilization of the two enzymes on different carriers and at different ratios of enzyme activities are described. When an inhibitor of catalase activity, such as NaN 3 or H 2O 2, is present, the conversion rate to 7-ACA is higher, but more by-products are obtained. An optimum ratio of 60:1 between the enzymatic activities of DAAO and Gl-7-ACA acylase in the coimmobilized sample at 0.21 Ug −1 Gl-7-ACA acylase activity was determined. The results of using coimmobilized enzymes and of using a mixture of separately immobilized enzymes in the same process are compared.

Application of enzyme flow microcalorimetry to the study of microkinetic properties of immobilized biocatalyst

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.

Permeabilization of Escherichia coli cells for enhanced penicillin acylase activity

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 8.0 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 0.2%, 20 min and 5°C respectively. Subsequently, the cell bound activity was retained for a longer period by chemical cross-linking with 0.1% glutaraldehyde.

Chemical modification and site-directed mutagenesis of tyrosine residues in cephalosporin C acylase from Pseudomonas strain N176

Cephalosporin C (CC) acylase from Pseudomonas strain N176 was chemically modified by tetranitromethane (TNM), causing complete loss of activity. Modification using molar excesses of TNM up to 10 resulted in complete inactivation when 1.4 mol tyrosines/mol enzyme were modified. Digestion of native and TNM-modified acylase with Achromobacter protease I (API), separation by high performance liquid phase chromatography (HPLC) and amino terminal sequencing of the resultant peptides were used to identify the modified tyrosine residues. The major difference in HPLC profile between these API digests was shown to be the peak corresponding to the peptide Ser 239-Lys 301 of native acylase. A portion of the peak for the peptide Ala 45-Lys 73 was also shifted in HPLC analysis of TNM-modified acylase. The peptides isolated from the modified acylase were shown to contain nitrated tyrosines (3-nitrotyrosine) at positions 270 and 52, respectively. These findings indicate that Tyr 270 is completely modified, and Tyr 52 is partially modified in the inactivated acylase. Each of the fifteen tyrosines in the acylase was altered to leucine by site-directed mutagenesis to complement the chemical modification with TNM. At pH 8.7, the mutant acylase in which tyrosine at position 270 is changed to leucine showed GL-7ACA and CC acylase activities reduced to 28.0 and 32.2% of native acylase, respectively. The results correspond to those obtained from TNM-modification. A similar reduction in activity was also obtained in the case of Tyr 491 mutant, although nitration of this residue was not confirmed by chemical modification. Therefore Tyr 270 and Tyr 491 are important for exerting the maximum activity of the enzyme, but are not essential for catalysis. However, mutation of Tyr 52 to Leu produced little change in acylase activity. The mutant acylase in which Tyr 705 is changed to leucine has a lowered pH optimum for GL-7ACA, which may be useful for further improvement of the acylase.

Kinetic study of penicillin acylase production by recombinant E. coli in batch cultures

The pPA102 plasmid, containing the penicillin acylase gene (pac) under the regulation of the lacZ gene promoter was constructed and used to transform E. coli JM101. Batch cultures of the recombinant strain were performed to characterize the kinetics of growth, substrate consumption, and penicillin acylase (PA) production under different induction conditions. A saturation type behaviour of PA activity with respect to the inducer (isopropyl-β-thio-galactopyranoside (IPTG) concentration was observed. No detrimental effect of recombinant protein expression was noted on growth rate, maximum protein and cell concentration, and glucose specific consumption rate. A simplification of an existing mechanistic kinetic model was used to describe the specific PA production rate, which exhibited a maximum at intermediate growth rates. Induction during inoculation resulted in the highest penicillin acylase activity compared to induction during later growth phases. Accumulation of PA precursor protein suggests that postranslational processing and translocation through the cytoplasmic membrane limit PA production.

Design and kinetic characterization of a whole cell penicillin acylase biocatalyst using E. coli

The design and characterization of a biocatalyst with whole cells of E. coli containing penicillin acylase activity were carried out. The biocatalyst was prepared by gel entrapment using a two-phase dispersion system consisting of a suspension of cells and agar in oil. 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. An economical analysis of the process was taken as the basis for a rational design of a genetic engineering programme to improve the specific activity of the biocatalyst.

Isolation and Characterization of a Pseudomonas Strain Producing Glutaryl-7-Aminocephalosporanic Acid Acylase

Several screening methods were developed for the selection of Pseudomonas strains capable of hydrolyzing glutaryl-7-aminocephalosporanic acid to 7-aminocephalosporanic acid. An isolate exhibiting high acylase activity, designated BL072, was identified as a strain of Pseudomonas diminuta. It grew optimally at pH 7 to 8 and at a temperature of 32 to 40 degrees C, but acylase activity was highest when the strain was grown at 28 degrees C. Mutants of BL072 were generated by nitrosoguanidine treatment and screened for increased production of glutaryl-7-aminocephalosporanic acid acylase. A superior mutant gave a fourfold increase in acylase titer. The cell-associated acylase had similar activities against various glutaryl-cephems but had undetectable activity against cephalosporin C. This acylase may prove useful for the conversion of cephalosporin C to 7-aminocephalosporanic acid.

Nephrotoxic and Genotoxic N-Acetyl-S-Dichlorovinyl-L-Cysteine Is a Urinary Metabolite after Occupational 1,1,2-Trichloroethene Exposure in Humans: Implications for the Risk of Trichloroethene Exposure

Excretion of mercapturic acids in the urine is indicative of the formation of electrophiles in the metabolism of xenobiotics. The determination of these mercapturic acids thus may be a useful method to estimate the exposure. We identified the nephrotoxic and mutagenic mercapturic acids N-acetyl-S-(1,2-dichlorovinyl)-L- cysteine and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine in the urine of workers exposed to 1,1,2-trichloroethene. A method to quantify these mercapturic acids by gas chromatography-mass spectrometry-selected ion monitoring was developed and appreciable amounts (2.8-3.8 mumole/L were found in human urine samples. Because deacetylation determines notably the amount of the excreted mercapturic acids, the formation of the resulting cysteine S-conjugates was comparably measured in subcellular fractions of rodent and human kidneys; significant species differences in acylase activity were found. The formation of mutagenic and nephrotoxic metabolites during 1,1,2-trichloroethene metabolism mandates a revision of the risk assessment of trichloroethene exposure.

Nephrotoxic and genotoxic N-acetyl-S-dichlorovinyl-L-cysteine is a urinary metabolite after occupational 1,1,2-trichloroethene exposure in humans: implications for the risk of trichloroethene exposure.

Excretion of mercapturic acids in the urine is indicative of the formation of electrophiles in the metabolism of xenobiotics. The determination of these mercapturic acids thus may be a useful method to estimate the exposure. We identified the nephrotoxic and mutagenic mercapturic acids N-acetyl-S-(1,2-dichlorovinyl)-L- cysteine and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine in the urine of workers exposed to 1,1,2-trichloroethene. A method to quantify these mercapturic acids by gas chromatography-mass spectrometry-selected ion monitoring was developed and appreciable amounts (2.8-3.8 mumole/L were found in human urine samples. Because deacetylation determines notably the amount of the excreted mercapturic acids, the formation of the resulting cysteine S-conjugates was comparably measured in subcellular fractions of rodent and human kidneys; significant species differences in acylase activity were found. The formation of mutagenic and nephrotoxic metabolites during 1,1,2-trichloroethene metabolism mandates a revision of the risk assessment of trichloroethene exposure.

Regulation of the supramolecular structure and the catalytic activity of penicillin acylase from Escherichia coli in the system of reversed micelles of Aerosol OT in octane

The properties of penicillin acylase from E. coli solubilized by hydrated reversed micelles (RM) of Aerosol OT in octane were studied. The dependence of catalytic activity on the hydration degree, a parameter which determines the size of the micelle inner cavity, has a curve with three optima, each one corresponding to the enzyme functioning either in a dimer form (w o = 23) or in a form of separate subunits, a heavy one, β, and a light one, α (w o = 20 and 14, respectively). The reversible dissociation of the enzyme was confirmed by ultracentrifugation followed by electrophoresis.