Purification and characterization of a superoxide dismutase from Panax ginseng

A superoxide dismutase (SOD) with the molecular weight of 31,079 has been purified as a homodimer from Panax ginseng by employing neutral pH buffer extraction, ammonium sulfate precipitation, isoelectric point precipitation and ion exchange methods. The enzyme's specific activity determined by an improved Marklund method was 9480.43 U/mg. Metal analysis showed that the SOD contained iron with the stoichiometry of 0.9 ± 0.3 Fe/subunit and exhibited high thermal stability (70°C) over the pH range from 4.0 to 9.0. Its maximum absorption wavelength was 278 nm and it was sensitive to hydrogen peroxide, trichloromethane‐ethanol and urea. These results indicate that the enzyme is an iron SOD. Copyright © 2010 John Wiley & Sons, Ltd.     

Purification and characterization of a nuclease from Lentinus edodes.

An endonuclease with 3'-nucleotidase activity (nuclease Le1) was purified from fruit bodies of Lentinus edodes in a single band on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The apparent molecular weight of nuclease Le1 was about 27000. The nuclease was inactivated in the presence of ethylenediaminetetraacetic acid (EDTA) and reactivated by the addition of Zn2+. Hydrolysis of poly U by the nuclease showed many intermediate size oligomers prior to the formation of 5'-uridine monophosphate (UMP). Therefore, it was concluded that nuclease Le1 was a Zn(2+)-endonuclease similar to P1-nuclease from Penicillium citrinum. The nuclease was very sensitive to ionic strength, but pH-profiles of the hydrolysis of four 3'-nucleotides were very similar to those of P1 nuclease from P. citrinum.     

Purification and characterization of hementin, a fibrinogenolytic protease from the leech Haementeria ghilianii

The fibrinogenolytic enzyme hementin, present in extracts of the posterior salivary glands of the giant leech Haementeria ghilianii, was isolated by ultrafiltration, high-performance ion-exchange chromatography and subsequent reversed-phase liquid chromatography. Approximately 100 micrograms (1 nmol) of hementin, present at less than 0.5% in the crude leech salivary extract, was brought to about 90% purity in three steps. Hementin migrated at an Mr of about 73,000 on non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and at 82,000 on reducing SDS-PAGE. The amino terminal sequence was determined to be TTLTE-PEPDL. The amino terminal sequences of two inactive proteins that partially coeluted with hementin in the first chromatographic step were also determined.     

Purification and characterization of dioscin-alpha-L-rhamnosidase from pig liver

Dioscin-alpha-L-rhamnosidase was isolated, purified and partially characterized from pig liver. The maximum activity was reached at pH 7, 42 degrees C, 24 h, and 2% of substrate concentration. Fe3+ and Cu2+ inhibited the enzyme; the ion Ca2+ activated it. Mg2+ was an inhibitor at 100 mM, but it was an activator at 200 mM. Zn2+ could be a weak activator of the enzyme. The molecular weight of dioscin-alpha-L-rhamnosidase was about 47 kDa as determined by the method of SDS-polyacrylamide gel electrophoresis.     

Purification and characterization of theD-hydantoinase from bacillus circulans

AD-hydantoinase (5,6-dihydropyrimidine amidohydrolase) was purified to homogeneity fromBacillus circulans. Purification of two hundred forty-three-fold was achieved with an overall yield of 12%. The relative molecular mass of the native enzyme is 212,000 and that of the subunit is 53,000. This enzyme is an acidic protein with an isoelectric point of 4.55. The enzyme is sensitive to thiol reagent and requires metal ions for its activity. The optimal conditions for the hydantoinase activity are pH 8.0–10.0 and a temperature of 75‡C. The enzyme is the most stable in a pH range of 8.5–9.5 and up to 60‡C. The enzyme is significantly stable not only at high temperatures but also on treatment with protein denaturant SDS. These remarkable properties are used for the purification procedure.     

Purification and characterization of ginsenoside-beta-glucosidase from ginseng.

The ginsenoside-beta-glucosidase that hydrolyzes the beta-(1-->2)-glucoside of the ginsenoside Rg3 sugar moiety to ginsenoside Rh2 was isolated from the ginseng root, and the enzyme was purified and characterized. The enzyme was purified to one spot in SDS polyacrylamide gel electrophoresis, and its molecular weight was about 59 kDa. The optimum temperature of the ginsenoside-beta-glucosidase was 60 degrees C, and the optimum pH was 5.0. Ca2+ ion had positive effect on ginsenoside-beta-glucosidase, while Cu2+ had negative effect on it. The ginsenoside-beta-glucosidase may be a special beta-glucosidase that is different from the original exocellulase such as beta-glucosidase (EC 3.2.1.21).     

Purification and characterization of a microbial dehydrogenase

Pseudomonas fluorescens (strain BTP9) was found to have at least two NAD(P)-dependent vanillin dehydrogenases: one is induced by vanillin, and the other is constitutive. The constitutive enzyme was purified by ammonium sulfate fractionation, gel-filtration, and Q-Sepharose chromatography. The subunit Mr value was 55,000, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native M _r value estimated by gelfiltration chromatography gave a value of 210,000. The enzyme made use of NAD⁺ less effectively than NADP⁺. Benzaldehyde, 4-hydroxybenzaldehyde, hexanal, and acetaldehyde were not oxidized at detectable rates in the presence of NAD⁺ or NADP⁺. The ultraviolet absorption spectrum indicated that there is no cofactor or prosthetic group bound. The vanillin oxidation reaction was essentially irreversible. The pH optimum was 9.5 and the pI of the enzyme was 4.9. Enzyme activity was not affected when assayed in the presence of salts, except FeCl₂. The enzyme was inhibited by the thiol-blocking reagents 4-chloromercuribenzoate and N-ethylmaleimide. NAD⁺ and NADP⁺ protected the enzyme against such a type of inhibition along with vanillin to a lesser extent. The enzyme exhibited esterase activity with 4-nitrophenyl acetate as substrate and was activated by low concentrations of NAD⁺ or NADP⁺. We compared the properties of the enzyme with those of some well-characterized microbial benzaldehyde dehydrogenases.     

Purification and characterization of a new Bacillus thuringiensis bacteriocin active against Listeria monocytogenes, Bacillus cereus and Agrobacterium tumefaciens

This study reports on the identification, characterization and purification of a new bacteriocin, named Bacthuricin F103, from a Bacillus thuringiensis strain BUPM103. Bacthuricin F103 production began in the early exponential phase and reached a maximum in the middle of the same phase. Two chromatographic methods based on high performance liquid chromatography and fast protein liquid chromatography systems were used to purify Bacthuricin F103. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that this bacteriocin had a molecular weight of approximately 11 kDa. It also showed a wide range of thermostability of up to 80 °C for 60 min and a broad spectrum of antimicrobial activity over a pH range of 3.0–10.0. This bacteriocin was noted, and for the first time, to exhibit potent antimicrobial activity against Agrobacterium subsp. strains, the major causal agents of crown gall disease in tomato and vineyard crops, and against several challenging organisms in food, such as Listeria monocytogenes and Bacillus cereus. Complete killing with immediate impact on cells was observed within a short period of time. The sequence obtained for Bacthuricin F103 by direct N-terminal sequencing shared considerable homology with hemolysin. Bacthuricin F103 was noted to act through the depletion of intracellular ions, which suggest that the cell membrane was a possible target to Bacthuricin F103.     

Purification and partial characterization of a lipase from Bacillus coagulans ZJU318

An extracellular lipase was purified from the fermentation broth of Bacillus coagulans ZJU318 by CM-Sepharose chromatography, followed by Sephacryl S-200 chromatography. The lipase was purified 14.7-fold with 18% recovery and a specific activity of 141.1 U/mg. The molecular weight of the homogeneous enzyme was (32 kDa), determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The enzyme activity was maximum at pH 9.0 and was stable over a pH range of 7.0–10.0, and the optimum temperature for the enzyme reaction was 45°C. Little activity loss (6.2%) was observed after 1 h of incubation at 40°C. However, the stability of the lipase decreased sharply at 50 and 60°C. The enzyme activity was strongly inhibited by Ag⁺ and Cu²⁺, whereas EDTA caused no inhibition. SDS, Brij 30, and Tween-80 inhibited lipase, whereas Triton X-100 did not significantly inhibit lipase activity.     

Purification and characterization of a laccase from the white-rot fungus Trametes multicolor

The wood-degrading fungus Trametes multicolor secretes several laccase isoforms when grown on a simple medium containing copper in the millimolar range for stimulating laccase synthesis. The main isoenzyme laccase II was purified to apparent homogeneity from the culture supernatant by using anion-exchange chromatography and gel filtration. Laccase II is a monomeric glycoprotein with a molecular mass of 63 kDa as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis, contains 18% glycosylation, and has a pI of 3.0. It oxidizes a variety of phenolic substrates as well as ferrocyanide and iodide. The pH optimum depends on the substrate employed and shows a bell-shaped pH activity profile with an optimum of 4.0 to 5.0 for the phenolic substrates, while the nonphenolic substrates ferrocyanide and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate) show a monotonic pH profile with a rate decreasing with increasing pH.     

Purification and characterization of a solvent stable protease from Pseudomonas aeruginosa PseA

A solvent tolerant Pseudomonas aeruginosa PseA strain was isolated from soil. It secreted a novel alkaline protease, which was stable and active in the presence of range of organic solvents, thus potentially useful for catalysis in non-aqueous media. The protease was purified 11.6-fold with 60% recovery by combination of ion exchange and hydrophobic interaction chromatography using Q-Sepharose and Phenyl Sepharose 6 Fast Flow matrix, respectively. The apparent molecular mass based on the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was estimated to be 35,000 Da. The enzyme was stable in the pH range of 6.0-9.0, the optimum being 8.0. The Km and Vmax towards caseinolytic activity were found to be 2.7 mg/ml and 3 micromol/min, respectively. The protease was most active at 60 degrees C and characterized as a metalloprotease because of its sensitivity to EDTA and 1,10-phenanthroline. It was tested positive for elastase activity towards elastin-orcein, thus appears to be an elastase, which is known as pseudolysin in other strains of P. aeruginosa. The protease withstands range of detergents, surfactants and solvents. It is stable and active in all the solvents having log P above 3.2, at least up to 72 h. These two properties make it an ideal choice for applications in detergent formulations and enzymatic peptide synthesis.     

Isolation and characterization of crotosparsamide, a new cyclic nonapeptide from Croton sparsiflorus

Crotosparsamide (1), a new cyclic nonapeptide, has been isolated from the n-butanol soluble sub-fraction of Croton sparsiflorus along with p-hydroxy methylcinnamate and kaempferol, which are reported for the first time from this species. Their structures were determined by chemical and spectral studies including ESIMS, and 1D and 2D NMR spectroscopic data.     

Purification and characterization of a nuclease (3'-nucleotidase) from a Penicillium sp

A nuclease (3'-nucleotidase) similar to P1 nuclease from Penicillium citrinum was purified from a commercial digestive from a Penicillium sp. The activity of the nuclease (PA) was separated to three fractions by diethylaminoethyl-Toyopearl 650M column chromatography, in total yield of 10%. The apparent molecular weight of these three nucleases, PA1, PA2 and PA3 was 35000, 33000, and 32000, respectively. All of them were homogeneous so far as checked by sodium dodecyl sulfate slab gel electrophoresis. The three nucleases differed in carbohydrate content, but their amino acid composition was practically the same, and very similar to that of P1 nuclease. The molecular weight of nuclease PA3, the major component of nuclease PA, was approximately 27000 after digestion by endoglycosidase F. The N-terminal and C-terminal amino acid sequences of nuclease PA3 were determined by Edman degradation and carboxypeptidase(s) digestion, respectively. The nuclease PA3 was inactivated in the presence of 10 mM ethylenediamine tetraacetic acid (EDTA) and 65% of its native enzyme activity restored by the addition of 20 mM ZnCl2. The pH-dependent photooxidative inactivation of nuclease PA3 was accelerated by removal of Zn ion by EDTA or trishydroxymethyl aminomethane, indicating the possible chelation of Zn2+ with some histidine residues.     

Purification and characterization of a cytolytic protein from purple fluid of the sea hare, Dolabella auricularia

A novel cytolytic factor, dolabellanin P, was purified to apparent homogeneity from the purple fluid of the sea hare, Dolabella auricularia. Purified dolabellanin P is a single polypeptide of 60 kDa. The amino acid composition and the N-terminus of the factor were also determined. This factor nonspecifically lysed all the cells tested at 50-200 ng protein/ml. Dolabellanin P caused complete cytolysis within 2 h. The factor is distinct from antineoplastic glycoproteins previously isolated from eggs (aplysianin E) or albumen gland (aplysianin A) of Aplysia kurodai in terms of certain cytolytic properties. These results suggest that dolabellanin P, found in the sea hare, a marine invertebrate, is a new cytolytic factor.     

Purification,characterization, and modification of T lymphocyte-stimulating polysaccharide from spores of Ganoderma lucidum

The hot-water extract of the spores of Ganoderma lucidum was shown to have a stimulating effect on concanavalin A-induced mitogenic activity of T lymphocytes. Bioassay-guided separation led to the isolation of a polysaccharide with potent T lymphocyte-stimulating activity by ethanol fractionation, anion-exchange, and size-exclusion chromatography. Based on the composition and methylation analyses, periodate oxidation, Smith degradation, and NMR spectroscopy, the native polysaccharide was shown to be a beta-D-(1-->3)-glucan with branches of terminal glucosyl residues substituted at C-6 of the glucose residues in the main chain. The branching ratio is approximately 20%. A series of sulfated or carboxymethylated derivatives were prepared and their structural features were elucidated by chemical and spectral analyses. The solution conformation and T lymphocyte proliferation effect of the glucans before and after derivatization were compared and discussed. The data obtained indicate that the introduction of ionic groups would significantly affect the original conformation of the native glucan in aqueous solution and further affect T lymphocyte-stimulating activity. The triple-helical structure of the glucans, the nature of the ionic groups, and the density of negative charge were considered to be closely related to this activity.     

Purification and characterization of an exoinulinase from Aspergillus fumigatus

An extracellular exoinulinase was purified from the crude extract of Aspergillus fumigatus by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-Sephacel, Sephacryl S-200, concanavalin A-linked amino-activated silica, and Sepharose 6B columns. The enzyme was purified 25-fold, and the specific activity of the purified enzyme was 171 IU/mg of protein. Gel filtration chromatography revealed a molecular weight of about 200 kDa, and native polyacrylamide gel electrophoresis (PAGE) showed an electrophoretic mobility corresponding to a molecular weight of about 176.5 kDa. Sodium dodecyl sulfate-PAGE analysis revealed three closely moving bands of about 66, 62.7, and 59.4 kDa, thus indicating the heterotrimeric nature of this enzyme. The purified enzyme appeared as a single band on isoelectric focusing, with a pI of about 8.8. The enzyme activity was maximum at pH 5.5 and was stable over a pH range of 4.0–9.5, and the optimum temperature for enzyme activity was 60°C. The purified enzyme retained 35.9 and 25.8% activities after 4 h at 50 and 55°C, respectively. The inulin hydrolysis activity was completely abolished with 1 mM Hg⁺⁺, whereas EDTA inhibited about 63% activity. As compared to sucrose, stachyose, and raffinose, the purified enzyme had lower K _m (0.25 mM) and higher V _max (333.3 IU/mg) values for inulin.