Efficient two-step chromatographic purification of penicillin acylase from clarified Escherichia coli ultrasonic homogenate

A two-step chromatographic purification procedure from clarified Escherichia coli ultrasonic homogenate was evaluated. The capture step included immobilized metal affinity chromatography with Cu²⁺ as metal ion. Two elution methods were performed: 1 M NH₄Cl and 0.01 M imidazole. Respectively, we obtained a different purification fold (16.5 to 3.15) and a similar result for the recovery of activity (90–99%). The best elution method was chosen for the procedure. The second step, hydrophobic interaction chromatography, gave a 3.8-fold purification with 77.7% of activity. The total procedure gave a 66-fold purification in relation to the initial crude extract with 70% for the recovery of activity and was performed without any conditioning step and at the same pH value.     

Optimization of a pseudo-affinity process for penicillin acylase purification

A pseudo-affinity process for penicillin acylase (EC 3.5.1.11) purification using an affinity ligand (Ampicillin) attached on Sepharose 4B-CNBr was optimized. The enzyme adsorption on this affiant (Amp-Seph) is independent of pH between 5.5 and 8.8, in 100?mM phosphate containing 22% (w/v) ammonium sulphate. The desorption of the penicillin acylase from the affinity gels was carried out, the best desorption results being obtained through a non specific eluent, 100?mM phosphate pH 4.6 with 15% (w/v) ammonium sulphate. The best purification results were obtained with an enzymatic extract, produced through osmotic shock of Escherichia coli cells (3.7?IU/mg prot). With this extract and an affinity gel of Sepharose 4B-CNBr derivatized with ampicillin (3.8?μmol/cm³?gel), a maximum activity capacity adsorbed of 20?IU/cm³?gel was obtained for initial values of activity and protein concentration of 1.7?IU/cm³ and 0.4?mg prot/cm³, respectively. With the optimized eluent it was possible to obtain penicillin acylase in only one purification step with a desorption yield of enzyme activity higher than 90%. The penicillin acylase produced with this process was characterized by a maximum purity of 34?IU/mg prot, corresponding to a purification degree higher than 150 in relation to the lowest pure enzymatic extract. The enzyme purity of the eluted fractions was certified by SDS gel electrophoresis and liquid chromatography through a Mono Q column in a FPLC apparatus. The gel electrophoresis presented 4 main stained bands with 2 corresponding to α and β subunits of the penicillin acylase with equivalent molecular weights of 27 and 63?kDa. No external diffusion resistance on penicillin acylase and total protein adsorption on this affiant (Amp-Seph 3.8?μmol/cm³?gel) were observed for continuous adsorption processes performed at two different agitation speeds (120 and 400?rpm).     

Preliminary studies on continuous recovery and purification of the penicillin acylase under pseudo-affinity conditions using phenyl-Sepharose gel

A continuous system for the recovery and purification of the penicillin acylase from crude extracts by recycling phenyl-Sepharose gel through three agitated vessels with disc filters of stainless steel is presented. The penicillin acylase present in the crude extract was absorbed into the phenyl-Sepharose gel under pseudo-affinity conditions (16% w/v of ammonium sulphate). After gel washing under the same conditions in the second vessel, enzyme desorption was performed using the same salt but at a lower concentration (6% w/v) in the third vessel. The preliminary studies reported here occurred without experimental difficulties, even at a gel concentration as high as 40% (v/v). The recovery of the penicillin acylase was achieved with high yield (74%), but a low purification factor of 2.4 was obtained owing to the use of a crude extract with low specific activity.     

Liquid–liquid extraction of antimicrobial peptide P34 by aqueous two-phase and micellar systems

Antimicrobial peptide P34 is a promising biopreservative for utilization in the food industry. In this work, aqueous biphasic systems (ABS) and aqueous biphasic micellar systems (ABMS) were studied as prestep for purification of peptide P34. The ABS was prepared with polyethylene glycol (PEG) and inorganic salts and the ABMS with Triton X-114 was chosen as the phase-forming surfactant. Results indicate that peptide P34 partitions preferentially to PEG-rich phase and extraction with ammonium sulfate [(NH₄)₂SO₄], yielding a 75% recovery of the antimicrobial activity, specific activity of 1,530 antimicrobial units per mg of protein, and purification fold of 2.48. Protein partition coefficient and partition coefficient for the biological activity with (NH₄)₂SO₄ system were 0.48 and 64, respectively. Addition of sodium chloride did not affect recovery, but decreased protein amount in the PEG-rich phase, indicating a higher partition of biomolecules. ABMS did not yield good recovery of antimicrobial activity. Purification fold using PEG–(NH₄)₂SO₄ and 1.0?mol l^?1 sodium chloride was twice higher than that obtained by conventional protocol, indicating a successful utilization of ABS as a step for purification of peptide P34.     

Purification and characterization of YxeI, a penicillin acylase from Bacillus subtilis

The paper reports the purification and characterization of the first penicillin acylase from Bacillus subtilis. YxeI, the protein annotated as hypothetical, coded by the gene yxeI in the open reading frame between iol and hut operons in B. subtilis was cloned and expressed in Eshcherichia coli, purified and characterized. The purified protein showed measurable penicillin acylase activity with penicillin V. The enzyme was a homotetramer of 148 kDa. The apparent K_m of the enzyme for penicillin V and the synthetic substrate 2-nitro-5-(phenoxyacetamido)-benzoic acid was 40 mM and 0.63 mM, respectively, and the association constants were 8.93 × 10² M⁻¹ and 2.51 × 10⁵ M⁻¹, respectively. It was inhibited by cephalosporins and conjugated bile salts, substrates of the closely related bile acid hydrolases. It had good sequence homology with other penicillin V acylases and conjugated bile acid hydrolases, members of the Ntn hydrolase family. The N-terminal nucleophile was a cysteine which is revealed by a simple removal of N-formyl-methionine. The activity of the protein was affected by high temperature, acidic pH and the presence of the denaturant guanidine hydrochloride.     

Effects of NH<Subscript>4</Subscript>CL application and removal on astrocytes and endothelial cells

Background

Hepatic encephalopathy (HE) is a complex disorder associated with increased ammonia levels in the brain. Although astrocytes are believed to be the principal cells affected in hyperammonemia (HA), endothelial cells (ECs) may also play an important role by contributing to the vasogenic effect of HA.

Methods

Following acute application and removal of NH₄Cl on astrocytes and endothelial cells, we analyzed pH changes, using fluorescence imaging with BCECF/AM, and changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i), employing fluorescence imaging with Fura-2/AM. Using confocal microscopy, changes in cell volume were observed accompanied by changes of [Ca²⁺]_i in astrocytes and ECs.

Results

Exposure of astrocytes and ECs to 1 – 20 mM NH₄Cl resulted in rapid concentration-dependent alkalinization of cytoplasm followed by slow recovery. Removal of the NH₄Cl led to rapid concentration-dependent acidification, again followed by slow recovery. Following the application of NH₄Cl, a transient, concentration-dependent rise in [Ca²⁺]_i in astrocytes was observed. This was due to the release of Ca²⁺ from intracellular stores, since the response was abolished by emptying intracellular stores with thapsigargin and ATP, and was still present in the Ca²⁺-free bathing solution. The removal of NH₄Cl also led to a transient concentration-dependent rise in [Ca²⁺]_i that resulted from Ca²⁺ release from cytoplasmic proteins, since removing Ca²⁺ from the bathing solution and emptying intracellular Ca²⁺ stores did not eliminate the rise. Similar results were obtained from experiments on ECs. Following acute application and removal of NH₄Cl no significant changes in astrocyte volume were detected; however, an increase of EC volume was observed after the administration of NH₄Cl, and EC shrinkage was demonstrated after the acute removal of NH₄Cl.

Conclusions

This study reveals new data which may give a more complete insight into the mechanism of development and treatment of HE.

     

Immobilization of penicillin G acylase on paramagnetic aldehyde-functionalized mesostructured cellular foams

Paramagnetic aldehyde-functionalized mesostructured cellular foams (PAMCFs), synthesized by grafting 3-aminopropyltriethoxysilane modified Fe₃O₄ (NH₂-Fe₃O₄) nanoparticles with larger particle size than the window pore size of MCFs on the outer surface of aldehyde-functionalized mesostructured cellular foams (AMCFs), were investigated as efficient supports for immobilization of penicillin G acylase (PGA). The results show that NH₂-Fe₃O₄ nanoparticles were successfully grafted on the outer surface of AMCFs and PGA molecules were mainly immobilized covalently on the inner surface of PAMCFs, which was because amino groups of NH₂-Fe₃O₄ nanoparticles or PGA molecules reacted with aldehyde groups of AMCFs or PAMCFs to form imine bonds. PGA/PAMCFs-15 showed a rather high initial activity of 9563 U g⁻¹ and retained 89.1% of its initial activity after recycled for 10 times. PGA/PAMCFs are easily recycled by magnetic field in order to replace tedious separation of high-speed centrifugation for mesoporous materials.     

Penicillin acylase purification with the aid of hydrophobic charge induction chromatography

The aim of this work was to test a chromatographic support, 4-mercaptoethyl pyridine (4-MEP) Hypercel, for penicillin acylase purification by using pure penicillin acylase and crude extract. Two equilibration buffers with various salt concentrations and different flow rates were tested. The relationships between electrostatic and hydrophobic interactions and proteins are demonstrated. (NH4)2SO4 proved preferable because no salting-in occurred, contrary to NaCl. The recovery and purification fold were similar to those obtained in pseudo-affinity chromatography with a three-fold reduction of the (NH4)2SO4 concentration.     

Biosynthesis of penicillin V acylase by Fusarium sp.: effect of culture conditions

Penicillin V acylase was produced, both intracellularly and extracellularly, by Fusarium sp. SKF 235 grown in submerged fermentation. When neopeptone was added to the medium, >95% of the penicillin V acylase was extracellular. In the absence of a complex organic nitrogen source, the fungus produced low levels of totally intracellular penicillin V acylase. MgSO₄ was essential for synthesis of the enzyme, which was induced by phenoxyacetic acid and penicillin V. The maximum yield of penicillin V acylase was 430 IU/g dry cell wt. The optimum pH value and temperature for the penicillin V acylase were 6.5 and 55°C, respectively.     

Contributions to nitrogen leaching and pasture uptake by autumn-applied dairy effluent and ammonium fertilizer labeled with 15N isotope

The objective of this study was to compare the N leaching loss and pasture N uptake from autumn-applied dairy shed effluent and ammonium fertilizer (NH₄Cl) labeled with ¹⁵N, using intact soil lysimeters (80 cm diameter, 120 cm depth). The soil used was a sandy loam, and the pasture was a mixture of perennial ryegrass (Lolium perenne) and white clover (Trifolium repens). The DSE and NH₄Cl were applied twice annually in autumn (May) and late spring (November), each at 200 kg N ha^-1. The N applied in May 1996 was labeled with ¹⁵N. The lysimeters were either spray or flood irrigated during the summer. The autumn-applied DSE resulted in lower N leaching losses compared with NH₄Cl. However, the N applied in the autumn had a higher potential for leaching than N applied in late spring. Between 4.5–8.1% of the ¹⁵N-labeled mineral N in the DSE and 15.1–18.8% of the ¹⁵N-labeled NH₄Cl applied in the autumn were leached within a year of application. Of the annual N leaching losses in the DSE treatments (16.0–26.9 kg N ha^-1), a fifth (20.3–22.9%) was from the mineral N fraction of the DSE applied in the autumn, with the remaining larger proportion from the organic fraction of the DSE, soil N and N applied in spring. In the NH₄Cl treatments, more than half (53.8–64.8%) of the annual N leaching loss (55.9–57.6 kg N ha^-1) was derived from the autumn-applied NH₄Cl. DSE was as effective as NH₄Cl in stimulating pasture production. Since only 4.4–4.5% of the annual herbage N uptake in the DSE treatment and 12.3–13.3% in the NH₄Cl treatment were derived from the autumn-applied mineral N, large proportions of the annual herbage N uptake must have been derived from the N applied in spring, the organic N fraction in the DSE, soil N and N fixed by clover. The recoveries of ¹⁵N in the herbage were similar between the DSE and the NH₄Cl treatments, but those in the leachate were over 50% less from the DSE than from the NH₄Cl treatment. The lower leaching loss of ¹⁵N in the DSE treatment was attributed to the stimulated microbial activities and increased immobilization following the application of DSE. This revised version was published online in June 2006 with corrections to the Cover Date.     

Purification of penicillin G acylase using immobilized metal affinity membranes

The immobilized metal affinity membrane (IMAM) with modified regeneration cellulose was employed for purification of penicillin G acylase (PGA). For studying PGA adsorption capacity on the IMAM, factors such as chelator surface density, chelating metal, loading temperature, pH, NaCl concentration and elution solutions were investigated. The optimal loading conditions were found at 4 degrees C, 0.5 M NaCl, 32.04 micromol Cu(2+) per disk with 10 mM sodium phosphate buffer, pH 8.5, whereas elution conditions were: 1 M NH(4)Cl with 10 mM sodium phosphate buffer, pH 6.8. By applying these chromatographic conditions to the flow experiments in a cartridge, a 9.11-fold purification in specific activity with 90.25% recovery for PGA purification was obtained. Meanwhile, more than eight-times reusability of the membrane was achieved with the EDTA regeneration solutions.     

Optimization of cell growth and poly(3-hydroxybutyrate) accumulation on date syrup by a Bacillus megaterium strain

Optimal growth and PHB accumulation in Bacillus megaterium occurred with 5% (w/v) date syrup or beet molasses supplemented with NH₄Cl. When date syrup and beet molasses were used alone without an additional nitrogen source, a cell density of about 3gl^–1 with a PHB content of the cells of 50% (w/w) was achieved. NH₄NO₃ followed by ammonium acetate and then NH₄Cl supported cell growth up to 4.8gl^–1, whereas PHB accumulation was increased with NH₄Cl followed by ammonium acetate, NH₄NO₃ and then (NH₄)₂SO₄ to a PHB content of nearly 42% (w/w). Cultivation of B.megaterium at 30l scale gave a PHB content of 25% (w/w) of the cells and a cell density of 3.4gl^–1 after 14h growth.     

Responses of salivary acinar cells to intracellular alkalinization

Responses of rat submandibular acini to intracellular alkalinization were investigated. Intracellular alkalinization was induced by addition of NH⁴Cl or methylamines, or by prepulse with Na butyrate. Only partial recovery occurred following Na butyrate prepulse or methylated amine addition, but full recovery was observed following addition of NH⁴Cl. The latter recovery was DIDS and dimethylamiloride-insensitive but was inhibited by bumetanide or high [K⁺] and stimulated in Na⁺ free buffer and by ouabain. Acetylcholine stimulated recovery from NH⁴Cl- or Na butyrate pre-pulse-induced alkalinization and reduced the extent of alkalinization induced by methylated amines. Acetylcholine-stimulated recovery from NH⁴Cl-induced alkalinization was mimicked by substance P or ionomycin and was partially Ca²⁺-dependent. This stimulated recovery was bumetanide-insensitive but was partially sensitive to charybdotoxin. Taken together, these data indicate that in unstimulated cells, recovery from alkalinization induced by NH⁴Cl occurs by bumetanide-sensitive transport of the NH⁴⁺ ion, that DIDS-inhibitable anion transport contributes little to this recovery, and that acetylcholine and other Ca²⁺-elevating agents accelerate recovery from NH⁴Cl-induced alkaline challenge by a mechanism insensitive to bumetanide, DIDS, ouabain, and dimethylamiloride but sensitive to extracellular Ca²⁺ and to charybdotoxin. Partial recovery from alkaline challenge can also occur in the absence of NH⁴⁺ ions, and acetylcholine also stimulates this mode of recovery. Together, these data suggest that these cells have little intrinsic ability to recover from intracellular alkalinization and that the NH⁴⁺ ion may be a surrogate for K⁺ in at least two ion transport pathways. © 1994 wiley-Liss, Inc.     

Effect of ammonium on nitrate utilization by roots of dwarf bean

The effect of exogenous NH₄⁺ on NO₃⁻ uptake and in vivo NO₃⁻ reductase activity (NRA) in roots of Phaseolus vulgaris L. cv Witte Krombek was studied before, during, and after the apparent induction of root NRA and NO₃⁻ uptake. Pretreatment with NH₄Cl (0.15-50 millimolar) affected neither the time pattern nor the steady state rate of NO₃⁻ uptake.

When NH₄⁺ was given at the start of NO₃⁻ nutrition, the time pattern of NO₃⁻ uptake was the same as in plants receiving no NH₄⁺. After 6 hours, however, the NO₃⁻ uptake rate (NUR) and root NRA were inhibited by NH₄⁺ to a maximum of 45% and 60%, respectively.

The response of the NUR of NO₃⁻-induced plants depended on the NH₄Cl concentration. Below 1 millimolar NH₄⁺, the NUR declined immediately and some restoration occurred in the second hour. In the third hour, the NUR became constant. In contrast, NH₄⁺ at 2 millimolar and above caused a rapid and transient stimulation of NO₃⁻ uptake, followed again by a decrease in the first, a recovery in the second, and a steady state in the third hour. Maximal inhibition of steady state NUR was 50%. With NO₃⁻-induced plants, root NRA responded less and more slowly to NH₄⁺ than did NUR.

Methionine sulfoximine and azaserine, inhibitors of glutamine synthetase and glutamate synthase, respectively, relieved the NH₄⁺ inhibition of the NUR of NO₃⁻-induced plants. We conclude that repression of the NUR by NH₄⁺ depends on NH₄⁺ assimilation. The repression by NH₄⁺ was least at the lowest and highest NH₄⁺ levels tested (0.04 and 25 millimolar).

     

Solubilization and reconstitution of the oat root vacuolar h/ca exchanger

Calcium is sequestered into vacuoles of oat (Avena sativa L.) root cells via a H⁺/Ca²⁺ antiporter, and vesicles derived from the vacuolar membrane (tonoplast) catalyze an uptake of calcium which is dependent on protons (pH gradient [ΔpH] dependent). The first step toward purification and identification of the H⁺/Ca²⁺ antiporter is to solubilize and reconstitute the transport activity in liposomes. The vacuolar H⁺/Ca²⁺ antiporter was solubilized with octylglucoside in the presence of soybean phospholipids and glycerol. After centrifugation, the soluble proteins were reconstituted into liposomes by detergent dilution. A ΔpH (acid inside) was generated in the proteoliposomes with an NH₄Cl gradient (NH₄⁺_in » NH₄⁺_out) as determined by methylamine uptake. Fundamental properties of ΔpH dependent calcium uptake such as the K_m for calcium (~15 micromolar) and the sensitivity to inhibitors such as N,N′-dicyclohexylcarbodiimide, ruthenium red, and lanthanum, were similar to those found in membrane vesicles, indicating that the H⁺/Ca²⁺ antiporter has been reconstituted in active form.     

Some aspects of chemical ionization mass spectroscopy using ammonia as reagent gas: A valuable technique for biomedical and natural products studies

The use of ammonia as reagent gas increases considerably the utility of chemical ionization mass spectroscopic (ci-ms) analysis: compounds of biological interest, such as steroid hormones, bile acids, prostaglandins, phospholipids, alkaloids, antibiotics, etc., display strong pseudomolecular ions (mostly M⁺ + 18). The need for derivatization and/or chromatographic purification of many types of compounds is sharply reduced. Ammonium carbonate or ¹⁵NH₄Cl can be introduced into the direct probe for obtaining satisfactory ci-ms(NH₃) spectra. Bile salts and some bile acid conjugates can be studied without derivatization. Potassium penicillanate gives a strong peak corresponding to the free acid + NH₄⁺. Deproteinized blood samples provide a detailed picture of individual components, such as triglycerides, lysolecithins, cholesterol esters, etc. Frag-mentation patterns for structural information can be generated by adding argon to ammonia. One shortcoming of the ci-ms(NH₃) method is the progressive replacement of halogen with hydrogen.     

Interaction of hypochlorous acid and myeloperoxidase with phosphatidylserine in the presence of ammonium ions

The close association of the heme enzyme myeloperoxidase to phosphatidylserine epitopes on the surface of non-vital polymorphonuclear leukocytes (PMNs) and other apoptotic cells at inflammatory sites favours modifications of this phospholipid by myeloperoxidase products. As detected by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, ammonium ions inhibit in a concentration-dependent manner the hypochlorous acid-mediated formation of aldehyde and nitrile products from 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS). Concomitantly, the formation of monochloramine (NH₂Cl) raises with increasing NH₄⁺ concentrations. A transchlorination from monochlorinated O-phospho-l-serine to NH₄⁺ with the formation of NH₂Cl occurs only when extraordinary high NH₄⁺ concentrations are applied. Due to the low rate of 0.044 M^− 1 s^− 1 for this process, a transhalogenation reaction from transient chlorinated intermediates of the serine moiety to NH₄⁺ can be ruled out as an important process contributing to the HOCl-mediated formation of NH₂Cl. A significant formation of NH₂Cl by myeloperoxidase interacting with DPPS in the presence of ammonium ions takes only place at acidic pH values around 5, a scenario that may occur in phagosomes of macrophages after the uptake of apoptotic PMNs.     

Regulation of cation transport pathways and glycolytic enzyme activity by alterations in red cell volume

In the presence of NH₄Cl and hypotonic solutions, Rana balcanica red cells respond by increasing their volume. The stimulation of cellular volume by hypotonicity is more rapid than that of NH₄Cl, while the maximum value is less than that observed in the presence of NH₄Cl. Depending on the cause of swelling, (net uptake of NH₄Cl or decrease in external osmolality) cells show specific responses. The NH₄Cl treatment causes a significant increase in intracellular Na⁺, from 5·14±0·78 to 29·84±0·47 mmoles l⁻¹ cell, while hypotonicity leads to a significant decrease of this cation, to 3·85±0·25 mmoles l⁻¹ cell in relation to the control, after 30 min of incubation of Rana balcanica erythrocytes. In addition, amiloride significantly reverses the NH₄Cl effect with respect to intracellular Na⁺. Both treatments cause a significant K⁺ loss in comparison with controls. Two glycolytic enzymes glyceraldehyde phosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) of Rana balcanica haemolysate were found to respond to the NH₄Cl effect by significantly decreasing their activity. Copyright © 1999 John Wiley & Sons, Ltd.     

Synthesis and characterization of pseudo-affinity ligand for penicillin acylase purification

The aim of this work was to test a chromatographic affinity support containing methacryloyl antipyrine (MAAP) for penicillin acylase (PA) purification by using pure penicillin acylase and crude extract. First, MAAP as a pseudo-specific ligand was synthesized by using methacryloyl chloride and 4-aminoantipyrine. Polymer beads (average size diameter: 40–120 μm) were prepared by suspension polymerization of ethylene glycol dimethacrylate (EGDMA) and MAAP. This approach for the preparation of adsorbent has several advantages over conventional preparation protocols. An expensive and time consuming step in the preparation of adsorbent is immobilization of a ligand to the adsorption matrix. In this procedure, affinity ligand MAAP acts as comonomer without further modification steps. Poly(EGDMA-MAAP) beads were characterized by FTIR, NMR and screen analysis. Elemental analysis of MAAP for nitrogen was estimated as 89.3 μmol/g. The prepared adsorbent was then used for the capture of penicillin acylase in batch system. The maximum penicillin acylase adsorption capacity of the poly(EGDMA-MAAP) beads was found to be 82.2 mg/g at pH 5.0. Chromatography with crude feedstock resulted in 23.2-fold purification and 93% recovery with 1.0 M NaOH.     

Effects of Ammonium and Potassium Ions on Some Physiological and Biochemical Processes of Excised Cucumber Cotyledons

Incubation of excised cucumber cotyledons (Cucumis sativus L.) with NH₄Cl solutions exceeding 0.001 M inhibited their greening, fresh weight increases, and incorporation of ¹⁴C-leucine into insoluble N compounds. The absorption of ¹⁴C-leucine during incubation and retention of moisture by the excised cotyledons after incubation were greatly diminished by the NH₄Cl treatments. Treatment with KCl solutions of the same concentrations as the NH₄Cl stimulated the greening, fresh weight increases, and the absorption and incorporation of ¹⁴C-leucine. Desiccation of cotyledons stored at 5°C for 10 days was inhibited by dilute KCl solutions. The toxicity of NH₄Cl was alleviated by KCl solutions at low concentrations.