Susceptibility of Rhodobacter sphaeroides to beta-lactam antibiotics: isolation and characterization of a periplasmic beta-lactamase (cephalosporinase).

Thirteen strains of the gram-negative, facultative phototrophic bacterium Rhodobacter sphaeroides were examined fro susceptibility to beta-lactam antibiotics. All strains were sensitive to the semisynthetic penicillins ampicillin, carbenicillin, oxacillin, cloxacillin, and methicillin, but 10 of the 13 strains were resistant to penicillin G, as well as a number of cephalosporins, such as cephalothin, cephapirin, and cephalosporin C. A beta-lactamase (EC 3.5.2.6) with strong cephalosporinase activity was detected in all of the resistant strains of R. sphaeroides. With strain Y-1 as a model, it was shown that the beta-lactamase was inducible by penicillin G, cephalosporin C, cephalothin, and to some minor extent, cephapirin. The beta-lactamase was located in the periplasmic space, from which it could be extracted by osmotic shock disruption. By using this fraction, the beta-lactamase was purified 34-fold to homogeneity by steps involving batch adsorption to and elution from DEAE-Sephadex A50, chromatography on Q-Sepharose, and preparative polyacrylamide gel electrophoresis. The molecular masses of the native and denatured enzymes were determined to be 38.5 kilodaltons by gel filtration and 40.5 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating a monomeric structure. The isoelectric point was estimated to be at pH 4.3. In Tris hydrochloride buffer, optimum enzyme activity was measured at pH 8.5. The beta-lactamase showed high activity in the presence of the substrates cephalothin, cephapirin, cephalosporin C, and penicillin G, for which the apparent Km values were 144, 100, 65, and 110 microM, respectively. Cephalexin, cepharidine, and cephaloridine were poor substrates. The beta-lactamase was strongly inhibited by cloxacillin and oxacillin but only slightly inhibited by phenylmethylsulfonyl fluoride or thiol reagents such as iodoacetate and p-chloromercuribenzoate.     

Purification and some properties of beta-lactamases from Proteus rettgeri and Proteus inconstans

Two beta-lactamases were isolated from strains of Proteus species and purified, one from a strain of P. rettgeri and the other from a strain of P. inconstans. Each enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis. Molecular weights of P. rettgeri and P. inconstans enzymes were found to be 42,000 and 43,000, and their isoelectric points pH 8.7 and 8.6, respectively. The two enzymes presented typical cephalosporinase profiles. Cefmetazole (CS-1170) and cefoxitin, both cephamycin antibiotics, not only resisted hydrolysis by both of the enzymes, but also inhibited their activities competitively. Rabbit antiserum against purified P. rettgeri enzyme inhibited the activity of both purified and crude enzyme preparations from other strains of P. rettgeri so far tested. None of the beta-lactamases produced by other species of Proteus including P. inconstans was inhibited by the antiserum, thus showing that the purified cephalosporinase was of the species-specific types. The enzymological properties of the preparations were compared with those of beta-lactamases derived from other gram-negative enteric bacteria.     

Biochemical Properties of a Cephalosporin β-Lactamase from Pseudomonas aeruginosa

The cephalosporin β-lactamase from Pseudomonas aeruginosa GN918 was purified using CM-Sepha-dex column chromatography. The resulting preparation gave a single protein peak on electrofocusing column chromatography and a single protein band on polyacrylamide-gel electrophoresis. The specific enzyme activity was 22 950 units per mg of the purified enzyme protein. The optimal pH was 7.5 and the optimal temperature was 40 C for the hydrolysis of cephaloridine. Isoelectric point was 8.7 and the approximate molecular weight of the enzyme was found to be 34 000±2000. The enzyme activity was inhibited by iodine, p-chloromercuribenzoate and semi-synthetic penicillins. The enzymological properties of the isolated preparation have been compared with β-lactamases derived from other gram-negative enteric bacteria.     

Penicillin-binding proteins in Proteus species.

Penicillin-binding proteins in three species of Proteus, Proteus mirabilis, P. morganii, and P. rettgeri, were investigated by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Penicillin-binding proteins in these Proteus species were compared with those in Escherichia coli K-12. An approximate correlation between penicillin-binding proteins in E. coli and those in Proteus species was shown by several criteria: electrophoretic mobilities; affinities of several beta-lactam antibiotics which show characteristic patterns of binding to penicillin-binding proteins in E. coli; relation between affinities of antibiotics to the proteins and effects on morphological changes in Proteus species; location of beta-lactamase activity among penicillin-binding proteins; and thermostability. The electrophoretic mobilities and several other characteristics of penicillin-binding proteins among the Proteus species examined were found to be similar from species to species and differed only slightly from those of E. coli.     

Induction of beta-lactamase in Proteus vulgaris

Various beta-lactam antibiotics, including monocyclic beta-lactams, induced the beta-lactamase of Proteus vulgaris; when clinical isolates were induced by benzylpenicillin, each strain produced a single beta-lactamase but the activity per milligram dry weight differed from strain to strain. The beta-lactamases of the P. vulgaris strains were heterogeneous with respect to their isoelectric points, but had almost the same specific activities, substrate specificities and Michaelis constants. The kinetics of beta-lactamase formation were investigated in three strains, each with a different beta-lactamase activity. Differential rates of enzyme synthesis and peak activity depended on the concentration of inducer. The plots of the reciprocals of the differential rates versus the reciprocals of the inducer concentrations were linear, and the maximum rate of enzyme synthesis and the concentration of the inducer giving half-maximum induction were determined from this double reciprocal plot. The maximum rates of enzyme synthesis were different in the three strains. The kinetic analysis of beta-lactamase formation revealed that the beta-lactamase activities in a single bacterial species were determined by differences in the rate of enzyme synthesis and not by differences in the properties of the enzyme.     

Purification and partial characterization of α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinofuranosidase produced by<Emphasis Type="Italic">Thermomonospora fusca</Emphasis>

Thermomonospora fusca produced a relatively high level of alpha-L-arabinofuranosidase when growing on oat spelt xylan as the main carbon and energy source. The enzyme exhibited maximum relative activity (0.136 U/g protein) at pH 9.0 with 54 and 55% activity remaining at pH of 4.5 and 11.0, respectively. The apparent Km value for the crude alpha-L-arabinofuranosidase preparation was 180 mumol/L 4-nitrophenyl alpha-L-arabinofuranoside; the upsilon lim value was the release of 40 mumol/L 4-nitrophenol per min. Enzyme activity was eluted as a single peak (HPLC gel filtration chromatography) corresponding to molar mass of approximately 92 kDa. Native electrophoresis of crude cell lysate confirmed the presence of a single active intracellular alpha-L-arabinofuranosidase component. SDS-PAGE of this enzyme, developed as zymogram, did not demonstrate any activity; denaturing gel was stained and a protein band of relative molar mass of 46 kDa was revealed. Isoelectric focusing of a purified alpha-L-arabinofuranosidase yielded a single protein band for the corresponding activity zone with pI 7.9. The enzyme was purified approximately 21-fold the mean overall yield was about 16%.     

Purification and Properties of Dextransucrase from Streptococcus mutans

The dextransucrase (EC 2.4.1.5) activity from cell-free culture supernatants of Streptococcus mutans strain 6715 has been purified approximately 1,500-fold by ammonium sulfate precipitation, hydroxylapatite chromatography, and isoelectric focusing. The enzyme was eluted as a single peak of activity from hydroxylapatite, and isoelectric focusing of the resulting preparation gave a single band of dextransucrase activity which focused at a pH of 4.0. The final enzyme preparation contained two distinct, enzymatically active proteins as judged by assay in situ after polyacrylamide gel electrophoresis. One of the proteins represented 90% of the total dextransucrase activity and 53% of the total protein. The molecular weight of the enzyme was estimated by gel filtration to be 94,000. The temperature optimum of the enzyme was broad (34 to 42 C) and its pH range was rather narrow, with optimal activity at pH 5.5. The K(m) for sucrose was 3 mM, and fructose competitively inhibited the enzyme reaction with a K(i) of 27 mM.     

Purification of pyruvate kinase from germinating castor bean endosperm

Cytosolic pyruvate kinase from endosperm of germinating castor beans (Ricinus communis L.; cv Hale) has been purified 3100-fold to apparent homogeneity and a final specific activity of 203 micromole pyruvate produced/minute per milligram protein. Purification steps included: heat treatment, polyethylene glycol fractionation, Q-Sepharose, ADP-agarose, Mono-Q and Phenyl Superose chromatography. Nondenaturing polyacrylamide gel electrophoresis of the final sample resulted in a single protein staining band which co-migrated with pyruvate kinase activity. Two protein staining bands of 57 and 56 kilodaltons were observed following SDS polyacrylamide gel electrophoresis of the final preparation. The native molecular mass was found to be about 240 kilodaltons. This enzyme appears to be a tetramer composed of two different subunits. The presence of dithioerythritol (2 millimolar) was required for optimal activity of the purified enzyme.     

Purification and some properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeniety from livers of thioacetamide- and dl-α-hydrazino-δ-aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5·10⁵-fold with an 8% yield; the specific activity of the final enzyme preparation was 1,1·10⁶ nmol CO₂/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105 000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50 000 on SDS-polyacrylmide gels. The isoelectric point of the enzyme was pH 4.1.     

Purification and characterization of glucose 6-phosphate dehydrogenase from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver

Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver, using a simple and rapid method, and some characteristics of the enzyme were investigated. The purification procedure was composed of two steps: homogenate preparation and 2', 5'-ADP Sepharose 4B affinity gel chromatography, which took 7-8 hours. Thanks to the two consecutive procedures, the enzyme, having specific activity of 38 EU/mg protein, was purified with a yield of 44.39% and 1310 fold. In order to control the enzyme purification SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. Optimal pH, stable pH, optimal temperature, Km and, Vmax values for NADP+ and glucose 6-phosphate (G6P) were also determined for the enzyme. In addition, molecular weight and subunit molecular weights were found by sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography respectively.     

Arylamidase of Cephalosporium acremonium and Its Specificity for Cephalosporin C

Three aggregational forms of arylamidase are produced by Cephalosporium acremonium. The exocellular enzyme, with an approximate molecular weight of 60,000, was purified 300-fold by diethylaminoethyl cellulose chromatography, gel filtration, and gel electrophoresis. With l-leucyl-beta-naphthylamide as the substrate, the K(m) is 4.2 x 10(-4)m; the optimum pH, 7.7; and the temperature optimum, 35 C. The enzymatic hydrolysis of l-leucyl-beta-naphthylamide is inhibited by a number of cephalosporins, whereas a variety of penicillins show no effect. Alternatively, the enzyme specifically catalyzes the beta-lactam hydrolysis of a number of cephalosporins; a number of penicillins are resistant. The K(m) for cephalosporin C is 9.09 x 10(-4)m.     

Isolation and characterization of a beta-lactamase-inhibitory protein from Streptomyces clavuligerus and cloning and analysis of the corresponding gene.

Culture filtrates of Streptomyces clavuligerus contain a proteinaceous beta-lactamase inhibitor (BLIP) in addition to a variety of beta-lactam compounds. BLIP was first detected by its ability to inhibit Bactopenase, a penicillinase derived from Bacillus cereus, but it has also been shown to inhibit the plasmid pUC- and chromosomally mediated beta-lactamases of Escherichia coli. BLIP showed no inhibitory effect against Enterobacter cloacae beta-lactamase, and it also showed no activity against an alternative source of B. cereus penicillinase. BLIP was purified to homogeneity, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a size estimate for BLIP of 16,900 to 18,000. The interaction between purified BLIP and the E. coli(pUC) beta-lactamase was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and determined to be noncovalent, with an estimated 1:1 molar stoichiometry. The BLIP gene was isolated on a 13.5-kilobase fragment of S. clavuligerus chromosomal DNA which did not overlap a 40-kilobase region of DNA known to contain genes for beta-lactam antibiotic biosynthesis. The gene encoded a mature protein with a deduced amino acid sequence of 165 residues (calculated molecular weight of 17,523) and also encoded a 36-amino-acid signal sequence. No significant sequence similarity to BLIP was found by pairwise comparisons using various protein and nucleotide sequence data banks or by hybridization experiments, and no BLIP activity was detected in the culture supernatants of other Streptomyces spp.     

Renal angiotensin I-converting enzyme as a mixture of sialo- and asialo-enzyme, and a rapid purification method.

Angiotensin I-converting enzyme [EC 3.4.15.1] was rapidly and highly purified from a particulate fraction of hog kidney cortex with 13% yield. The procedure, which was rapid, included fractionation on DEAE-cellulose and calcium phosphate gel, chromatographies on DEAE-Sephadex A-50 and hydroxylapatite columns, and gel filtration on a Sephadex G-200 column. The purified enzyme preparation gave two protein bands on standard disc gel electrophoresis, but showed a single protein component on the gel after treatment with neuraminidase [EC 3.2.1.18]. The data strongly suggest that the purified enzyme preparation was a mixture of sialo- and asialo-enzyme. Sialic acid residues apparently do not contribute to the catalytic activity of the enzyme. The enzyme was activated more by chloride ions than by other halide ions tested, using Bz-Gly-Gly-Gly as a substrate. The dissociation constant for chloride ions was determined to be 2.2 mM. Chloride did not protect the enzyme against heat or low pH. The enzyme was resistant to inactivation by trypsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1].     

Purification and properties of a novel thermostable galacto-oligosaccharide-producing beta-galactosidase from Sterigmatomyces elviae CBS8119.

A thermostable beta-galactosidase which catalyzed the production of galacto-oligosaccharide from lactose was solubilized from a cell wall preparation of Sterigmatomyces elviae CBS8119. The enzyme was purified to homogeneity by means of chromatography on DEAE-Toyopearl, Butyl-Toyopearl, Chromatofocusing, and p-aminobenzyl 1-thio-beta-D-galactopyranoside agarose columns. The molecular weight of the purified enzyme was estimated to be about 170,000 by gel filtration with a Highload-Superdex 200pg column and 86,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its isoelectric point, determined by polyacrylamide gel electrofocusing, was 4.1. The optimal temperature for enzyme activity was 85 degrees C. It was stable at temperatures up to 80 degrees C for 1 h. The optimal pH range for the enzyme was 4.5 to 5.0, it was stable at pH 2.5 to 7.0, and its activity was inhibited by Hg2+. The Km values for o-nitrophenyl-beta-D-galactopyranoside and lactose were 9.5 and 2.4 mM, respectively, and the maximum velocities for these substrates were 96 and 240 mumol/min per mg of protein, respectively. In addition, this enzyme possessed a high level of transgalactosylation activity. Galacto-oligosaccharides, including tri- and tetrasaccharides, were produced with a yield, by weight, of 39% from 200-mg/ml lactose.     

Purification and properties of N-acetylgalactosamine 6-sulphate sulphatase from human placenta

1. N-Acetylgalactosamine 6-sulphate sulphatase was purified about 20000-fold from the soluble extract of human placenta with N-acetylgalactosamine 6-sulphate-glucuronic acid-N-acetyl[1-(3)H]galactosaminitol 6-sulphate as substrate in the activity assay. The enzyme appears to be a glycoprotein with a mol.wt. of about 100000 as determined by gel filtration. On gel electrophoresis in the presence of sodium dodecyl sulphate the major protein band had a mol.wt. of 78000. Variable charge heterogeneity was observed in several enzyme preparations. 2. The purified enzyme released up to one sulphate molecule from the disulphated trisaccharide. It was active towards N-acetylgalactosamine 6-sulphate and exhibited no measurable N-acetylglucosamine 6-sulphate sulphatase or any other known lysosomal sulphatase activity. Hydrolysis of [1-(3)H]galactitol 6-sulphate was achieved by incubation neither with a crude nor with a purified enzyme preparation. Chondroitin 6-sulphate and keratan sulphate, as well as heparin and heparan sulphate, served as competitive inhibitors of the enzyme. 3. Purified N-acetylgalactosamine 6-sulphate sulphatase activity was optimal at pH4.9 and 4.4 when assayed in 0.02m-sodium acetate buffer and at pH4.2 and 5.2 in 0.1m-sodium acetate buffer. A single pH-optimum at pH4.8 was observed for the crude enzyme and for the purified enzyme after mild periodate treatment. The sulphatase activity was inhibited by a variety of anions and cations and activated by thiol-specific and thiol reagents.     

Purification and characterization of lysophospholipase released from rat platelets

Lysophospholipase released from rat platelets upon activation with thrombin has been purified to near homogeneity by sequential column chromatography on heparin-Sepharose, CM-Sephadex C-50, and TSK gel G2000SW. The final preparation showed a single band with a molecular mass of 32,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining. The purified enzyme was heat-labile and inactivated after 5 min at 60 degrees C. It showed a broad pH optimum (pH 6-10) and required a divalent cation, such as Ca2+, for the optimal activity. Appreciable activity, however, was observed in the presence of EDTA. Lysophospholipase activity was inhibited by diisopropylfluorophosphate and dithiothreitol. This enzyme activity was retained by a concanavalin A-Sepharose column and eluted with methyl-alpha-D-mannoside. Treatment of lysophospholipase with peptide: N-glycosidase F gave degraded products, suggesting that this protein contain N-linked carbohydrate chains. The purified enzyme was specific to 1-acyl-sn-glycero-3-phospho-L-serine; none of lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, and 1-acyl-sn-glycero-3-phospho-D-serine was hydrolyzed appreciably.     

Improved purification of α-amylase isolated fromRhizomucor pusillus by affinity chromatography

A highly purified extracellular -amylase was isolated fromRhizomucor pusillus with minimum loss of enzymatic activity. The enzyme was purified from the mycelium-free liquid filtrate of the thermophilic moldRhizomucor pusillus. Maximum enzyme yields were attained after 5 days of growth on liquid starch-yeast extract at 45°C and pH 7.0. The crude enzyme preparation was first concentrated 80-fold by ultrafiltration. Purification was recently achieved with high-performance liquid chromatography and Waters Protein Pak 300 SW. Improved purification was then achieved with a dextrin-bound affinity column, with a 59-fold increase in specific activity from the crude enzyme preparation. This final enzyme preparation produced a single band on polyacrylamide gel electrophoresis. The molecular weight determined by SDS gel electrophoresis was 52,000 daltons.     

Beta-lactamase activity of bacteria of the genus Proteus]

beta-Lactamases of Proteus and their role in the mechanism of the microbe resistance to penicillins and ceporin were studied. It was found that the beta-lactamase of Proteus had low activity and were produced by both beta-lactamide resistant and sensitive clinical strains of Proteus. The resistant cultures of Proteus produced enzymes more frequently (3.4--5 times) than the sensitive ones. The synthesis of beta-lactamase in the clinical Proteus strains was inducable. The high induction coefficient was achieved only in the presence of high concentrations of the inductor. No significant dependence of the culture sensitivity level of ampicillin and ceporin on the induction level was observed. The most significant part of the constitutive enzyme in Proteus was intracellular, while that of the inducable enzyme was extracellular. No correlative dependence between the culture resistance levels to penicillins and ceporin and the enzyme activity was noted. The beta-lactamase activity was not found in the transconjugants with the in vitro acquired R-factor controlling the ampicillin and ceporin resistance, as well as in the resistant mutants selected on the media with increasing concentrations of the above antibiotics. Induction of beta-lactamase synthesis was not found in these strains either. The ability of Proteus to synthesize beta-lactamase can be lost on the strain storage under laboratory conditions which was not always accompanied by reduction of the culture sensitivity to ampicillin and ceporin. The enzymatic destruction of beta-lactamides was not the main mechanism of Proteus resistance to the above antibiotics.     

Purification and properties of streptococcal nicotinamide adenine dinucleotide glycohydrolase.

Highly purified streptococcal nicotinamide adenine dinucleotide glycohydrolase (NADase) was obtained by utilizing disodium tetrathionate to protect the enzyme by blocking the sulfhydryl groups of streptococcal proteinase. This was followed by two-step ion-exchange chromatography. The pure enzyme, demonstrated as a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, had a specific activity of 11,200 NADase units per mg of protein and was devoid of hemolytic activity. NADase had a molecular weight of about 55,000 as determined by gel filtration, by summation of amino acid residues, and by sodium dodecyl sulfate/gel electrophoresis. The purified enzyme had optimal activity at pH 7.3 and at 40 C and a calculated Km of 5.1 times 10- minus 4 mM. It was inhibited by alpha-iodoacetamide.