Detection of extracellular proteinase of Pseudomonas fragi by enzyme-linked immunosorbent assay

A double-antibody-sandwich, enzyme-linked immunosorbent assay was developed to detect an extracellular proteinase produced by Pseudomonas fragi. The method was capable of detecting 4 g/ml of the proteinase in spiked samples of buffer and broth and 4.2 g/ml in a broth culture of the organism. The assay detected the presence of proteinase at bacterial densities of approximately 10⁴ cfu/ml, which develop after incubation for 15 h at 25°C in a broth medium. All assays could be completed within 7 h. This assay is of value in plotting proteolytic expression in relation to the growth cycle of Ps. fragi in broth culture and may be of value, with development, in other more complex milieux.     

Purification and characterization of an extracellular carbonic anhydrase from Pseudomonas fragi

Extracellular carbonic anhydrase was purified from Pseudomonas fragi isolated from CaCO₃ enriched soil samples. The enzyme is induced in presence of CaCO₃ and is envisaged to play an important role in bicarbonate ion transport. The 75% ammonium sulphate dialysate was purified by single step affinity chromatography with 86% yield. It is a trimeric protein having a subunit molecular weight of 31.0 kDa and was stable at pH 7.0–8.5 and temperature 35–45 °C. Lead, mercury and EDTA had an inhibitory effect on CA activity, whereas zinc, iron and cadmium increased it. The presence of esterase activity along with IC₅₀ of sulphonamides and anionic inhibitors indicated that CA from P. fragi belonged to α-class. The CA stability in presence of different salts, as well as in alkaline pH and high temperature makes it a potential candidate to be exploited for biomimetic CO₂ sequestration.     

Centroid search optimization of cultural conditions affecting the production of extracellular proteinase by Pseudomonas fragi ATCC 4973

The production of extracellular proteinase by Pseudomonas fragi ATCC 4973 grown in a defined citrate medium, containing glutamine as the sole nitrogen source, was determined under varying cultural conditions. Simultaneous evaluation of cultural conditions using a 'centroid search' optimization technique showed that the optimum cultural conditions for proteinase production by Ps. fragi were: incubation temperature, 12.5 degrees C; incubation time, 38 h; initial pH, 6.8; organic nitrogen concentration, 314 mmol nitrogen/l (glutamine); a gas mixture containing 16.4% oxygen flowing over the medium (7.42 ppm dissolved oxygen). Oxygen was the major factor influencing proteinase production by Ps. fragi. The results may have applications in the storage of fluid milk. Centroid search optimization was shown to be suitable for microbiological experiments.     

Centroid search optimization of cultural conditions affecting the production of extracellular proteinase by Pseudomonas fragi ATCC 4973

M yhara , R.M. & S kura , B. 1990. Centroid search optimization of cultural conditions affecting the production of extracellular proteinase by Pseudomonas fragi ATCC 4973. Journal of Applied Bacteriology 69 , 530–538.
The production of extracellular proteinase by Pseudomonas fragi ATCC 4973 grown in a defined citrate medium, containing glutamine as the sole nitrogen source, was determined under varying cultural conditions. Simultaneous evaluation of cultural conditions using a 'centroid search' optimization technique showed that the optimum cultural conditions for proteinase production by Ps. fragi were: incubation temperature, 12.5°C; incubation time, 38 h; initial pH, 6.8; organic nitrogen concentration, 314 mmol nitrogen/1 (glutamine); a gas mixture containing 16.4% oxygen flowing over the medium (7.42 ppm dissolved oxygen). Oxygen was the major factor influencing proteinase production by Ps. fragi . The results may have applications in the storage of fluid milk. Centroid search optimization was shown to be suitable for microbiological experiments.     

Isolation of an extracellular proteinase (keratinase) from Microsporum canis

Proteolytic activity was demonstrated when a strain of Microsporum canis was cultured in broth with human hair, but not in the medium without hair. The extracellular proteinase (keratinase) was isolated and purified by chromatography. Disc electrophoresis showed one protein band of extracellular proteinase, and the antibody against this enzyme gave a single precipitin line in agar diffusion. The IgG fraction completely neutralized the proteinase activity. The proteinase of M. canis may play a role in infection caused by this fungus, by affecting keratinized tissue such as stratum corneum and hair.     

Ultrastructural localization of an extracellular protease in Pseudomonas fragi by using the peroxidase-antiperoxidase reaction.

An extracellular protease, which previously has been found to correlate with the appearance of bleblike evaginations on the cell wall of Pseudomonas fragi ATCC 4973, was purified 38-fold by ammonium sulfate precipitation and Sephadex chromatography to yield a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyclonal rabbit antiserum raised against the purified enzyme had an enzyme-linked immunosorbent assay titer of 4 X 10(7). The peroxidase antiperoxidase method was used to localize the neutral protease in P. fragi at the ultrastructural level. Electron microscopy of cell sections of this organism revealed that high concentrations of positive immunoperoxidase reaction product were located near the cell wall, whereas control sections stained with preimmune or heterologous serum did not show similar deposits to be present. These results are consistent with the hypothesis that blebs appearing on P. fragi contain high concentrations of neutral protease.     

Ultrastructural localization of an extracellular protease in Pseudomonas fragi by using the peroxidase-antiperoxidase reaction

An extracellular protease, which previously has been found to correlate with the appearance of bleblike evaginations on the cell wall of Pseudomonas fragi ATCC 4973, was purified 38-fold by ammonium sulfate precipitation and Sephadex chromatography to yield a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyclonal rabbit antiserum raised against the purified enzyme had an enzyme-linked immunosorbent assay titer of 4 X 10(7). The peroxidase antiperoxidase method was used to localize the neutral protease in P. fragi at the ultrastructural level. Electron microscopy of cell sections of this organism revealed that high concentrations of positive immunoperoxidase reaction product were located near the cell wall, whereas control sections stained with preimmune or heterologous serum did not show similar deposits to be present. These results are consistent with the hypothesis that blebs appearing on P. fragi contain high concentrations of neutral protease.     

Isolation and properties of an extracellular proteinase of Staphylococcus aureus

Serine proteinase splitting the peptide bonds which are formed by carboxyl groups of dicarboxylic amino acids was isolated from the supernatant of the Staphylococcus aureus culture liquid. It is similar to the enzyme isolated by Drapeau from Staphylococcus aureus strain V8 in its specifidity, molecular weight, amino acid composition, existence of two pH optima (pH 4.6 and 8.2). But there are some differences between the two proteinase in the content of dicarbonic amino acid residues. It was found that the enzyme can exist in two molecular forms.     

Lipase from Pseudomonas fragi. I. Purification of the Enzyme

The experimental conditions required to isolate a lipase from Pseudomonas fragi were determined. The organism was grown in a buffered tryptone medium for 4 to 5 days at 20 C. The lipase in the culture supernatant fluid was isolated by fractionation with ammonium sulfate at 60% saturation, followed by acetone precipitation at 30-60% concentration. Further purification was made by using Sephadex G-200 gel-filtration and diethylaminoethyl cellulose chromatography. Electrophoretic analysis of the purified lipolytic fraction showed apparent homogeneity by both cellulose polyacetate and disc electrophoresis. The specific activity of the purified enzyme was about 100 times that of the starting culture filtrate, and the yield was about 1.8% of the original activity.     

Lipase from Pseudomonas fragi. II. Properties of the Enzyme

The optimal pH value of a lipase from Pseudomonas fragi was between 7.5 and 8.9, and a high reaction rate was observed at 54 C. Heating the enzyme solution at 63 C for 30 min inactivated only 27.6% of its activity; however, total inactivation was observed at 66 C after 1 hr and at 71 C after 10 min. The lipase was inhibited strongly by Fe⁺⁺⁺ and Fe⁺⁺ ions, and to a lesser extent by Co⁺⁺, Cu⁺⁺, Zn⁺⁺. No inhibition was observed with Ca⁺⁺ or NaF. Ethylenediaminetetraacetate was effective in removing the toxicity of Fe⁺⁺⁺. The activity of the enzyme was inhibited markedly by p-chloromercurobenzoate, but the effects of N-ethylmaleimide and iodoacetate were moderate. The enzyme was able to hydrolyze natural fats, synthetic triglycerides, and alcohol esters. The order of the rate of hydrolysis of some triglycerides under experimental conditions was, from the fastest to the lowest, trilaurin, tricaprin, tricaprylin, tripalmitin, tributyrin, tricaproin, and tristearin. The enzyme was capable of hydrolyzing methyl butyrate, but the rate of hydrolysis was about one-fifth that for triolein and one-thirteenth that for coconut oil. The enzyme lost its activity rapidly when held frozen, at 20 C, and at the extremes in pH. Glutathione, cysteine, and mercaptoethanol did not preserve the activity of the enzyme.     

Isolation and properties of neutral SH-dependent proteinase from bovine spleen]

A neutral SH-dependent proteinase was isolated from bovine spleen by a slight modification of the previous method (1) and its action on some natural and synthetic substrates was studied. The activity of the enzyme was increased 2000--2500-fold as compared to that of the original extract. The enzyme hydrolyzed various histones (H1, H2a, H2b, H3), casein and protamine but did not split hemoglobin, serum albumin and 14C-tryptophane-labelled total protein from chicken embryos. The enzyme possessed neither collagenolytic nor elastase activity; it did not inactivate aldolase, hexokinase, glyceraldehyde phosphate dehydrogenase and glucose-6-phosphate dehydrogenase, which makes the enzyme different from cathepsin B1 and some other previously described proteinases. The enzyme did not split BAPA, BAEE, ATEE, Boc-Ala-ONP, Leu-beta-NA and some other peptides. The molecular weight of the enzyme was found to be about 15 000.     

The structure and serologic distribution of an extracellular neutral polysaccharide from Pseudomonas aeruginosa immunotype 3

Previous work has described small molecular weight neutral polysaccharides from isolates of Pseudomonas aeruginosa that appear to be associated with the lipopolysaccharide (LPS) and distributed across serologic barriers defined by antibody to the O side chain. We have isolated and characterized another of these structures obtained from culture supernatants of an immunotype 3 strain of P. aeruginosa. The isolated neutral polysaccharide has a tetrasaccharide repeat unit: (formula; see text) where Rha is rhamnose. The structure was determined by 1H and 13C nuclear magnetic resonance spectroscopy including nuclear Overhauser enhancement experiments, acid hydrolysis, methylation analysis, Smith degradation, and optical rotation determinations. Polyclonal antibodies raised to intact and alkali-treated (0.1 N NaOH, 56 degrees C, 2 h) LPS from the seven Fisher immunotype strains of P. aeruginosa bound well to the neutral polysaccharide. Antibodies affinity purified from these sera using immobilized neutral polysaccharide as well as a neutral polysaccharide-specific monoclonal antibody, E87, reacted with an antigenically similar structure found among many isolates of different LPS serotypes in a colony blot and with LPS from the seven Fisher immunotypes in an immunoblot. In an immunoblot assay, the neutral polysaccharide inhibited binding of the monoclonal antibody, E87, to material present in LPS preparations from a variety of serotypes. This structure may represent another P. aeruginosa neutral polysaccharide variant found associated with the LPS.     

Neutral proteinase inhibitors in PMN leukocytes. II. Isolation and characterization of a neutral proteinase inhibitor from porcine PMN leukocytes

An inhibitor of neutral proteinases was purified from porcine PMN leukocytes by gel filtration on Sephadex G-75 superfine and ion-exchange chromatography on Mono S. Thus an inhibitor preparation with a specific inhibitory activity against chymotrypsin of 10 IU/mg was obtained. In dodecyl sulfate gel electrophoresis a single protein band with an apparent molecular mass of 40 kDa was found under reducing conditions. Under non-reducing conditions the inhibitor forms higher molecular mass aggregates. On isoelectric focusing several protein bands with isoelectric points between pH 7.0 and 7.5 could be separated. The amino-acid composition of the inhibitory protein was determined. The inhibition mechanism was studied and association rate constants (kon) were measured and calculated for the reaction with chymotrypsin as well as leukocyte and pancreatic elastase. In Western blot analysis and in enzyme immunoassay studies crossreactivity between antibodies directed against porcine leukocyte neutral proteinase inhibitor and the corresponding inhibitor of bovine PMN leukocytes could be demonstrated.     

Isolation and characterisation of a neutral proteinase from the canine intervertebral disc

A neutral proteinase of 94 kDa capable of degrading gelatin, canine disc proteoglycan, and L-lysine and L-arginine peptide substrates has been isolated from the greyhound intervertebral disc. Strong inhibition of this proteinase with class-specific inhibitors, such as APMSF, TLCK and benzamidine indicated a 'serine'-type specificity. Metallo, aspartyl- and cysteine proteinase inhibitors were devoid of significant action. Degradation of the resident canine disc proteoglycan monomer by the disc proteinase was shown to occur at the hyaluronic acid binding region, thereby diminishing its ability to aggregate with hyaluronic acid. The hydrodynamic size of the proteoglycan degradation products was only slightly less than that of the intact disc proteoglycan subunits.     

Isolation of an iron-binding compound from Pseudomonas aeruginosa.

An iron-binding compound was isolated from ethyl acetate extracts of culture supernatant fluids of Pseudomonas aeruginosa and was purified by successive paper and thin-layer chromatographic procedures. The purified compound was characterized by UV, visible, infrared, and fluorescence spectroscopy. The compound possesses phenolic characteristics, with little or no similarity to dihydroxybenzoates and no indication of a hydroxamate group. P. aeruginosa synthesized the compound during active growth in culture media containing less than 5 X 10(-6) M added FeCl3. When added to iron-poor cultures of P. aeruginosa, the compound promoted the growth of the bacterium and also reversed growth inhibition by the iron chelator ethylenediamine-di-(o-hydroxyphenylacetic acid).