An advanced dual labeled gold nanoparticles probe to detect Cryptosporidium parvum using rapid immuno-dot blot assay

The zoonotic protozoan parasite Cryptosporidium parvum poses a significant risk to public health. Due to the low infectious dose of C. parvum, remarkably sensitive detection methods are required for water and food industries analysis. However PCR affirmed sensing method of the causative nucleic acid has numerous advantages, still criterion demands for simple techniques and expertise understanding to extinguish its routine use. In contrast, protein based immuno detecting techniques are simpler to perform by a commoner, but lack of sensitivity due to inadequate signal amplification. In this paper, we focused on the development of a mere sensitive immuno detection method by coupling anti-cyst antibody and alkaline phosphatase on gold nanoparticle for C. parvum is described. Outcome of the sensitivity in an immuno-dot blot assay detection is enhanced by 500 fold (using conventional method) and visually be able to detect up to 10 oocysts/mL with minimal processing period. Techniques reported in this paper substantiate the convenience of immuno-dot blot assay for the routine screening of C. parvum in water/environmental examines and most importantly, demonstrates the potential of a prototype development of simple and inexpensive diagnostic technique.     

An immunoassay for detection of heat-stable proteases from thermoduric psychrotrophic Bacillus spp. of dairy origin

A homogeneous preparation of a thermostable protease from Bacillus sp. B-17 was used to raise an antiserum in rabbits. IgG of this antiserum was used to study the antigenic relationship of proteases in cell-free extracts of 21 bacilli of milk origin. Based on immunological cross reactivity, the 21 bacilli were divided into 3 serological subgroups. To raise antibodies of broader specificity, protease from Bacillus sp. B-11 (group II) and B-3 (group III) were purified, mixed with purified B-17 protease, and an antiserum was raised against this mixture. IgG of this antiserum was purified (IgG anti-bacilli protease). A sandwich ELISA was standardized using IgG anti-bacilli protease as capture antibody. The assay could detect 1.2 ng ml(-1) of protease in milk or buffer, but the assay failed to detect 4 of 21 bacilli proteases. The results suggest that this assay is useful for the detection of proteases of Bacillus spp. in dairy industry.     

An unusual thermostable aspartic protease from the latex of Ficus racemosa (L.)

The most extensively studied ficins have been isolated from the latex of Ficus glabrata and Ficus carica. However the proteases (ficins) from other species are less known. The purification and characterization of a protease from the latex of Ficus racemosa is reported. The enzyme purified to homogeneity is a single polypeptide chain of molecular weight of 44,500 ± 500 Da as determined by MALDI-TOF. The enzyme exhibited a broad spectrum of pH optima between pH 4.5-6.5 and showed maximum activity at 60 ± 0.5 °C. The enzyme activity was completely inhibited by pepstatin-A indicating that the purified enzyme is an aspartic protease. Far-UV circular dichroic spectra revealed that the purified enzyme contains predominantly β-structures. The purified protease is thermostable. The apparent T_m, (mid point of thermal inactivation) was found to be 70 ± 0.5 °C. Thermal inactivation was found to follow first order kinetics at pH 5.5. Activation energy (E_a) was found to be 44.0 ± 0.3 kcal mol⁻¹. The activation enthalpy (ΔH^∗), free energy change (ΔG^∗) and entropy (ΔS^∗) were estimated to be 43 ± 4 kcal mol⁻¹, −26 ± 3 kcal mol⁻¹ and 204 ± 10 cal mol⁻¹ K⁻¹, respectively. Its enzymatic specificity studied using oxidized B chain of insulin indicates that the protease preferably hydrolyzed peptide bonds C-terminal to glutamate, leucine and phenylalanine (at P₁ position). The broad specificity, pH optima and elevated thermal stability indicate the protease is distinct from other known ficins and would find applications in many sectors for its unique properties.     

Development of a fluorescent F-actin blot overlay assay for detection of F-actin binding proteins

Interactions between cellular proteins and filamentous (F) actin are key to many cellular functions, e.g., cell motility, endocytosis, cell:cell adhesion, and cell:substrate adhesion. Previously, a functional assay using 125I-labeled F-actin to detect a subset of F-actin binding proteins by blot overlay was developed. We have modified this assay to use the fluorescent label, Alexa 488, in place of 125Iodine. The detection limit for Alexa 488-labeled actin using a Molecular Dynamics STORM 860 Fluorescence/PhosphorImager was as little as 100pg of labeled actin. The Alexa 488 F-actin assay detects the same proteins from Dictyostelium discoideum and with approximately the same sensitivity (approximately 10 microg/ml F-actin final concentration) as the analogous 125I-labeled F-actin blot overlay. The use of Alexa 488 F-actin for blot overlay assays requires no radioactive materials and generates no hazardous waste. Assays can be performed on the laboratory bench top and the blots imaged directly with a blue laser scanner, either wet or dry. In addition, the Alexa 488 fluorophore is highly resistant to photobleaching, does not decay, and may be stored frozen or lyophilized. Alexa 488 F-actin is a stable, cost-effective, nonhazardous probe used for rapid identification of a subset of F-actin binding proteins.     

Production of extremely thermostable alkaline protease from Bacillus sp. no. AH-101

Summary An alkalophilic Bacillus sp. no. AH-101, which produced extremely thermostable alkaline protease, was isolated among 200 soil samples. The enzyme production reached its maximum level of 1500 units/ml after about 24 h in alkaline medium (pH 9.5). The enzyme was most active toward casein at pH 12–13 and stable to 10 min incubation at 60° C from pH 5–13. Calcium ions were effective in stabilizing the enzyme especially at higher temperatures. The optimum and stable temperatures were about 80° C and below about 70° C respectively in the presence of 5 mM calcium ions. The enzyme was completely inactivated by phenylmethane sulphonyl fluoride, but little affected by ethylenediaminetetraacetic acid, urea, sodium dodecylbenzenesulphonate and sodium dodecyl sulphate. The molecular weight and sedimentation constant were approximately 30 000 and 3.0S respectively, and the isoelectric point was at pH 9.2. These results indicte that no. AH-101 alkaline protease is more stable against both temperature and highly alkaline conditions than any other protease so far reported.     

A thermostable neutral protease from Pseudomonas aeruginosa CCRC 15541

S.-F. LU AND P.-P. CHANG. 1996. A bacterium producing thermostable neutral protease was isolated from an orchard in Taiwan and identified as Pseudomonas aeruginosa . Optimal activity of the protease from Ps. aeruginosa was observed at 60°C and pH 7.0. The protease was thermostable, 85% activity being retained after incubation at 60°C for 1 h. Enzyme activity could be inhibited by EDTA, indicating the protease might require a metal ion for its activity. The protease was active in the presence of surface-active reagents.     

Specific detection of the gene for the extracellular neutral protease of Bacillus cereus by PCR and blot hybridization.

A pair of primers and a gene probe for the amplification and detection of the Bacillus cereus neutral protease gene (NPRC) were developed. Specificity for the npr genes of the B. cereus group members B. cereus, B. mycoides, and B. thuringiensis was shown. Restriction polymorphism patterns of the PCR products confirmed the presence of the NPRC gene in all three species.     

Purification and characterization of two thermostable protease fractions from Bacillus megaterium

Protease enzyme from Bacillus megaterium was successively purified by ammonium sulfate precipitation, ion exchange chromatography on DEAE-cellulose and gel filtration chromatography on Sephadex G-200. The purification steps of protease resulted in the production of two protease fractions namely protease P1 and P2 with specific activities of 561.27 and 317.23 U mg^?1 of protein, respectively. The molecular weights of B. megaterium P1 and P2 were 28 and 25 KDa, respectively. The purified fractions P1 and P2 were rich in aspartic acid and serine. Relatively higher amounts of alanine, leucine, glycine, valine, thereonine valine and glutamic acid were also present. The maximum protease activities for both enzyme fractions were attained at 50 °C, pH 7.5, 1% of gelatine concentration and 0.5 enzyme concentrations. P1 and P2 fractions were more stable over pH 7.0–8.5 and able to prolong their thermal stability up to 80 °C. The effect of different inhibitors on the protease activity of both enzyme fractions was also studied. The enzyme was found to be serine active as it had been affected by lower concentrations of phenylmethylsulfonyl fluoride (PMSF). Complete dehairing of the enzyme-treated skin was achieved in 12 h, at room temperature.     

A nonradioactive dot blot assay for transglutaminase activity

Aberrant transglutaminase (TG) activity has been implicated in the pathology of numerous diseases, including Huntington’s disease and Alzheimer’s disease. To fully characterize the role of TGs in these disorders, it is important that simple quantifiable assays be made available. The most commonly used assay currently employed requires significant time and a radioactive substrate. The assay described here uses a biotinylated substrate in conjunction with a dot blot apparatus to eliminate the use of radioactive substrates and allows relative transglutaminase activity to be measured simultaneously with minimal sample preparation in a large number of samples containing purified enzyme, cell extracts, or tissue homogenates.     

An activity gel assay for the detection of DNA helicases and nucleases from cell-free extracts.

An activity gel assay was developed for the detection of DNA helicases in crude extracts. The assay was based on the ability of DNA helicases to unwind radioactive fragments from single-stranded M13 circles that were immobilized in an SDS polyacrylamide gel. The displaced radioactive strands were detected by blotting them to a filter and visualizing the resulting bands by autoradiography. Experiments with purified proteins demonstrated that DNA helicases, endonucleases and exonucleases could produce activity bands. A one-dimensional gel assay was sufficiently sensitive to allow detection of DNA helicase I, DNA helicase II, DNA helicase IV, the RecQ helicase as well as 3 unidentified putative DNA helicases in crude extracts of Escherichia coli. Exonuclease and endonuclease activities from crude extracts could be distinguished from DNA helicase activities by their ATP-independence and from each other by their band morphologies.     

Purification and characterization of a thermostable alkaline protease from alkalophilic Thermoactinomyces sp. HS682.

Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatographies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relative molecular mass of 25,000 according to gel filtration on a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0. Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DFP and PMSF, and metal ions, Cu2+ and Hg2+. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37 degrees C for 60 min. The optimum pH was pH 11.5-13.0 at 37 degrees C and the optimum temperature was 70 degrees C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5 mM CaCl2, it showed maximum proteolytic activity at 80 degrees C and stability from pH 4-12.5 at 60 degrees C and below 75 degrees C at pH 11.5. The stabilization by Ca2+ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of microbial serine protease, although alanine of the NH2-terminal amino acid was deleted.     

The role of bound calcium ions in thermostable, proteolytic enzymes. II. Studies on thermolysin, the thermostable protease from Bacillus thermoproteolyticus.

The functional properties of the four calcium ions, bound by thermolysin, appear to be very similar to those of the single calcium ion bound by thermomycolase (G. Voordouw and R.S. Roche (1975), Biochemistry, preceding paper in this issue). Hence when the free calcium ion concentration is varied in the range where the calcium double-site dissociates (G. Voordouw and R.S. Roche (1974), Biochemistry 13, 5017), no changes are observed in the sedimentation coefficient or the peptide circular dichroism. Differences in molar ellipticity and molar extinction coefficient occur in the aromatic ultraviolet region, which parallel the occupancy of the calcium binding double site. The difference spectrum, characterized by a main band at 290 nm and a somewhat smaller band at 283 nm, is interpreted as due to the transfer of a partially buried tryptophan residue to the aqueous solvent upon dissociation of the two calcium ions from the double site. This is most likely Trp-186, which is in between Asp-185 and Glu-187, two chelating amino acids of this site. From the calcium dependence of the rate constant for autolytic degradation we conclude, as for thermomycolase, that only conformers devoid of bound calcium ion serve as substrates in the reaction. This rate constant increases about 1000-fold, when the double site dissociates. Hydrogen-tritium exchange studies show the presence of a large stable strcutural core, comprising about 32% of all the peptide hydrogens present. These do not exchange-in after 24 hr at 25degreesC, pH 9.0, ionic strenth 0.1. The exchange-out of 60 slow hydrogens was found to be independent of the free calcium ion concentration in the range 2.0-8.0 X 10(-4) M, where all four calcium-binding sites are saturated. The calcium dependence of the first-order rate constant for thermal denaturation at 80degreesC, pH 7.0, indicates that thermolysin is stabilized by only one calcium ion under these conditions. These observations are rationalized in terms of a calcium-binding model for thermolysin and the known three-dimensional structure of the enzyme and its calcium-binding sites.     

Multicenter evaluation of reverse line blot assay for detection of drug resistance in Mycobacterium tuberculosis clinical isolates

A multicenter study was conducted with the objective to evaluate a reverse line blot (RLB) assay to detect resistance to rifampin (RIF), isoniazid (INH), streptomycin (STR), and ethambutol (EMB) in clinical isolates of Mycobacterium tuberculosis. Oligonucleotides specific for wild type and mutant (drug resistance linked) alleles of the selected codons in the genes rpoB, inhA, ahpC, rpsL, rrs, embB, were immobilized on a nylon membrane. The RLB assay conditions were optimized following analysis of DNA samples with known sequences of the targeted genes. For validation of the method at different geographical locations, the membranes were sent to seven laboratories in six countries representing the regions with high burdens of multudrug-resistant tuberculosis. The reproducibility of the assay for detection of rpoB genotypes was initially evaluated on a blinded set of twenty reference DNA samples with known allele types and overall concordant results were obtained. Further mutation analysis was performed by each laboratory on the local strains. Upon RLB analysis of 315 clinical isolates from different countries, 132 (85.2%) of 155 RIF-resistant and 28 (51.0%) of 55 EMB-resistant isolates were correctly identified, showing applicability of the assay when targeting the rpoB hot-spot region and embB306. Mutations in the inhA and ahpC promoter regions, conferring resistance to INH, were successfully identified in respectively 16.9% and 13.2% of INH-resistant strains. Likewise, mutations in rrs513 and rpsL88 that confer resistance to STR were identified in respectively 15.1% and 10.7% of STR-resistant strains. It should be mentioned that mutation analysis of the above targets usually requires rather costly DNA sequencing to which the proposed RLB assay presents rapid and inexpensive alternative. Furthermore, the proposed method requires the same simple equipment as that used for spoligotyping and permits simultaneous analysis of up to 40 samples. This technique is a first attempt to combine different targets in a single assay for prediction of antituberculosis drugs resistance. It is open to further development as it allows easy incorporation of new probes for detection of mutations in other genes associated with resistance to second-line (e.g., fluoroquinolones) and new antituberculosis compounds.     

Purification and characterization of a thermostable alkaline protease from alkalophilic Bacillus pumilus

AIMS: An investigation was carried out on the purification and characterization of an alkaline protease from Bacillus pumilus MK6-5. METHODS AND RESULTS: An alkalophilic Bacillus pumilus MK6-5 was grown in a laboratory fermenter containing 1% reverse osmosis concentrated cheese whey powder, 0.25% corn steep liquor, 1% glucose, 0.5% tryptone, 1% sodium citrate, 0.02% MgSO4.7H2O and 0.65% Na2CO3 at 35 degrees C and pH 9.6, agitation at 250 rev min(-1) and aeration of 1 vvm for 60 h. When the enzyme was purified using ammonium sulphate precipitation, ion exchange and gel filtration chromatographies, a 26.2% recovery of enzyme with 36.6-fold purification was recorded. The purified protease was found to be homogenous by SDS-PAGE with molecular mass estimate of 28 kDa. The enzyme was optimally active at pH 11.5 and temperature of 55-60 degrees C. The Km and kcat values observed with synthetic substrates at 37 degrees C and pH 8.0 were 1.1 mmol l(-1) and 624 s(-1) for Glu-Gly-Ala-Phe-pNA and 3.7 mmol l(-1) and 826 s(-1) for Glu-Ala-Ala-Ala-pNA, respectively. The kinetic data revealed that small aliphatic and aromatic residues were the preferred residues at the P1 position. Inhibition profile exhibited by PMSF suggested the B. pumilus protease to be an alkaline serine protease. CONCLUSIONS: Bacillus pumilus MK6-5 produced a calcium-dependent, thermostable alkaline serine protease. SIGNIFICANCE AND IMPACT OF THE STUDY: The thermostable alkaline protease from Bacillus pumilus MK6-5 will be extremely useful in ultrafiltration membrane cleaning due to its ability to work in broad pH and temperature ranges, and tolerance to detergents, unlike the mesophilic proteases which face these limitations.     

Multiplex PCR-based reverse line blot assay for simultaneous detection of 22 virulence genes in uropathogenic Escherichia coli

Urinary tract infections (UTIs) are among the most common bacterial infections and are responsible for significant morbidity and health care costs worldwide. The main bacterial cause of uncomplicated UTI is Escherichia coli, which possesses numerous virulence factors (VFs). Many studies of the pathogenesis of E. coli UTI have centered on VF genes. Hence, the development of better molecular assays to study VF genes would facilitate these studies. We developed a highly sensitive and specific multiplex PCR-based reverse line blot (mPCR/RLB) assay to simultaneously detect 22 VF genes of uropathogenic E. coli and then used it to characterize 180 isolates from nonpregnant women of child-bearing age with cystitis and 153 fecal isolates from similar-age healthy women, in regional New South Wales, Australia. The assay accurately identified all VF genes (of the 22 under study) known to be present in 30 previously characterized control strains. The detection limits were 28 ng of DNA from E. coli isolates and 50 CFU/ml in mock-infected urine specimens containing known concentrations of E. coli. Cystitis isolates (compared to the fecal isolates) showed a significantly higher prevalence of 18 individual VF genes and contained significantly more VF genes per isolate (median number, 18.5 versus 6.5 [P = 0.001]). Discordance between paired probes for a given VF gene occurred in several clinical test isolates but no reference strains and among the test isolates was associated with fecal source (10% of VF genes versus 2% for cystitis isolates [P < 0.001]). This novel mPCR/RLB method is a potentially powerful tool for investigating the prevalence and distribution of VFs in E. coli.     

Comparison of chemical assay, bioassay, enzyme-linked immunosorbent assay, and dot blot hybridization for detection of aerobactin in members of the family Enterobacteriaceae.

In order to determine the best strategy for detection of aerobactin in members of the family Enterobacteriaceae, we compared the results of three phenotypic assays, including a chemical assay, a cross-feeding bioassay, and an enzyme-linked immunosorbent assay (ELISA), with the results of a dot blot hybridization assay using a specific probe for the aerobactin genes. The sensitivity and specificity of the ELISA were better than those of the chemical and cross-feeding assays, but the results of dot blot hybridization were the most reproducible. However, none of the Serratia and Enterobacter cloacae strains which produced aerobactin hybridized with the probe. We concluded that the best strategy for aerobactin detection is a two-step procedure that combines screening by dot blot hybridization with an ELISA for negative strains.     

Enhanced production and characterization of a highly thermostable alkaline protease from Bacillus sp. P-2

An obligatory alkalophilic Bacillus sp. P-2, which produced a thermostable alkaline protease was isolated by selective screening from water samples. Protease production at 30 °C in static conditions was highest (66 U/ml) when glucose (1% w/v) was used with combination of yeast extract and peptone (0.25% w/v, each), in the basal medium. Protease production by Bacillus sp. P-2 was suppressed up to 90% when inorganic nitrogen sources were supplemented in the production medium. Among the various agro-byproducts used in different growth systems (solid state, submerged fermentation and biphasic system), wheat bran was found to be the best in terms of maximum enhancement of protease yield as compared to rice bran and sunflower seed cake. The protease was optimally active at pH 9.6, retaining more than 80% of its activity in the pH range of 7–10. The optimum temperature for maximum protease activity was 90 °C. The enzyme was stable at 90 °C for more than 1h and retained 95 and 37% of its activity at 99 °C and 121 °C, respectively, after 1 h. The half-life of protease at 121 °C was 47 min.     

Purification and characterization of a thermostable thiol protease from a newly isolated hyperthermophilic Pyrococcus sp.

A hyperthermophilic archaeon strain, KOD1, was isolated from a solfatara at a wharf on Kodakara Island, Kagoshima, Japan. The growth temperature of the strain ranged from 65 to 100 degrees C, and the optimal temperature was 95 degrees C. The anaerobic strain was an S0-dependent heterotroph. Cells were irregular cocci and were highly motile with several polar flagella. The membrane lipid was of the ether type, and the GC content of the DNA was estimated to be 38 mol%. The 16S rRNA sequence was 95% homologous to that of Pyrococcus abyssi. The optimum growth pH and NaCl concentration of the strain KOD1 were 7.0 and 3%, respectively. Therefore, strain KOD1 was identified as a Pyrococcus sp. Strain KOD1 produced at least three extracellular proteases. One of the most thermostable proteases was purified 21-fold, and the molecular size was determined to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45 kDa by gel filtration chromatography. The specific activity of the purified protease was 2,160 U/mg of protein. The enzyme exhibited its maximum activity at approximately pH 7.0 and at a temperature of 110 degrees with azocasein as a substrate. The enzyme activity was completely retained after heat treatment at 90 degrees C for 2 h, and the half-life of enzymatic activity at 100 degrees C was 60 min. The proteolytic activity was significantly inhibited by p-chloromercuribenzoic acid or E-64 but not by EDTA or phenylmethylsulfonyl fluoride. Proteolytic activity was enhanced threefold in the presence of 8 mM cysteine. These experimental results indicated that the enzyme was a thermostable thiol protease.     

Production of thermostable acid protease by Aspergillus niger

Aspergillus niger F2078 produces high levels of extracellular thermostable acid protease within 96 h. Although glucose and peptone were the best carbon and nitrogen sources, respectively, sucrose and a cheap nitrogen source, corn steep liquor, also gave satisfactory enzyme yields. Supplementation of groundnut meal to the basal medium enhanced enzyme production. Temperature and pH optima of the enzyme activity were 60°C and 3.0–4.0, respectively. The enzyme was stable between pH 3.0 and 6.0 and at temperatures up to 60°C.