Purification and Some Properties of Thiosulfate Dehydrogenase from Acidithiobacillus ferrooxidans

Abstract

Thiosulfate dehydrogenase was purified from Acidithiobacillus ferrooxidans using three purification steps. The purification procedure involved ammonium sulfate fractionation, ion‐exchange chromatography, and gel permeation chromatography. Specific activity of the purified enzyme (after IEC) was 3.26 nkat/mg, and yield of the enzyme was 78%. The purity of the enzyme was checked by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme is a tetramer composed of four probably identical subunits of relative molecular weight 45,000. The pH optimum of the enzyme reaction in the direction of substrate oxidation was found to be 3.0. The isoelectric point of the enzyme was 8.3. Enzyme activity was found to be particularly sensitive to the histidine‐selective reagent diethylpyrocarbonate. Reagents selective for arginine, cysteine, and tryptophane had no effect on enzyme activity.     

Purification and Characterization of S-Adenosyl-L-Methionine Nicotinic Acid-N-Methyltransferase from Leaves of Glycine max

Trigonelline (TRG), which act as a cell cycle regulator and a compatible solute in response to salinity and water-stress, is the N-methyl conjugate of nicotinic acid the formation of which is catalyzed by S-adenosyl-L-methionine nicotinic acid-N-methyltransferase. The enzyme was purified 2650-fold from soybean (Glycine max L.) leaves with a recovery of 4 %. The purification procedure included ammonium sulfate (45 – 60 %) precipitation, linear gradient DEAE-Sepharose chromatography, adenosine-agarose affinity chromatography, hydroxyapatite chromatography and gel filtration (Sephacryl-S-200). The purified enzyme preparation showed a major band with a molecular mass of 41.5 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis that is related to the enzyme activity. The native enzyme had a molecular mass of about 85 kDa as estimated by gel filtration. The K_m values for S-adenosyl-L-methionine and nicotinic acid were 31 and 12.5 M, respectively. The purified enzyme showed optimum activity at pH 6.5 and temperature of 40 – 45 °C. High concentration of dithiothreitol (10 mM) and glycerol (20 %) stabilize the enzyme during purification and storage. Hg²⁺ strongly inhibits enzyme activity.     

Purification and characterization of dye degrading of veratryl alcohol oxidase from Pseudomonas aeruginosa strain BCH

In the present study we have purified the intracellular veratryl alcohol oxidase (VAO) enzyme from Pseudomonas aeruginosa strain BCH to evaluate its dye decolorizing potential. The enzyme was purified by ion exchange chromatography using DEAE cellulose followed by gel filtration chromatography using Biogel P-100. The molecular weight of the purified enzyme was estimated by polyacrylamide gel electrophoresis (PAGE) analysis. The VAO was purified up to 12 and 16.3-fold by ion exchange and gel filtration chromatography respectively. VAO was estimated to be about 85 kDa by SDS–PAGE. The optimum pH and temperature for purified VAO was 3 and 55°C respectively. The purified enzyme exerted its optimal activity with veratryl alcohol and also oxidized various other substrates, whereas diminished activity was noted in case of tryptophan and xylidine. The metal ions Mn⁺⁺ and Hg⁺⁺ were found to suppress the oxidase activity. The purified enzyme decolorized different dyes with variable decolorization rates and efficiencies. Decolorization mechanism of Remazol Black by purified enzyme was studies in detail using various analytical techniques like HPLC, GC–MS and FTIR. This study is useful for understanding the precise role of Pseudomonas aeruginosa strain BCH in the decolorization of textile dyes containing industrial wastewater.     

Ferredoxin-dependent Nitrite Reductase from Spinach Leaves

A ferredoxin-dependent nitrite reductase from Spinacea oleracea was purified approximately 180-fold, with a specific activity of 285 units/mg protein. This purified enzyme also had methyl viologen-dependent nitrite reductase activity, with a specific activity of 164 units/mg protein. After disc electrophoresis with polyacrylamide gel, the purified enzyme showed one major and one minor protein band.

The molecular weight of the enzyme was estimated to be 86,000 from Ultrogel filtration. This purified enzyme in oxidized form had absorption peaks at 278, 390, 573 and 690 nm. The absorbance ratios, A₃₉₀: A₂₇₈ and A₆₇₃: A₃₉₀ were 0.61 and 0.37, respectively.

By applying the purified enzyme to DEAE-Sephadex A–50 column chromatography, the ferredoxin-dependent nitrite reductase activity was selectively decreased. However, the methyl viologen-dependent nitrite reductase activity was increased, with a specific activity of 391 units/mg protein. This modified enzyme was homogeneous by disc electrophoresis with polyacrylamide gel.     

Purification of Chlorpromazine-Sensitive GTPase from Rat Cerebral Cortex

Abstract

The chlorpromazine-sensitive GTPase from the cell membrane of rat cerebral cortex was purified to homogenity by using DEAE Bio-Gel A agarose, hydroxyapatite and heparin agarose chromatography. The purified chlorpromazine-sensitive GTPase was purified 370-fold to obtain a final specific activity of 40 nmol GTP hydrolyzed/min/mg protein. The purified enzyme was inhibited by chlorpromazine but not by compound 48/80. Magnesium was required for its activity instead of calcium. The purified enzyme had an apparent pH optimum of 8.0, and molecular weight was estimated to be 58,000.     

Rapid Purification of Yeast Cytoplasmic Fumarate Reductase Affinity Chromatography on Blue Sepharose CL–6B

Abstract

The rapid and effective purification of soluble fumarate reductase from baker's yeast achieved by Blue Sepharose CL–6B chromatography. Cibacron Blue F3GA, the chromophore of Blue Sepharose, inhibited the activity of fumarate reductase. The enzyme bound to the column was selectively eluted by flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) or riboflavin. The purified enzyme was essentially homogeneous as indicated by polyacrylamide gel electrophoresis under non-denaturing conditions and under denaturing conditions in sodium dodecylsulfate. By this procedure, the enzyme could be rapidly purified with high yield from yeast cells.     

Production, purification and characterization of an extracellular inulinase from Kluyveromyces marxianus var. bulgaricus

The yeast Kluyveromyces marxianus var. bulgaricus produced large amounts of extracellular inulinase activity when grown on inulin, sucrose, fructose and glucose as carbon source. This protein has been purified to homogeneity by using successive DEAE-Trisacryl Plus and Superose 6HR 10/30 columns. The purified enzyme showed a relative molecular weight of 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 77 kDa by gel filtration in Superose 6 HR 10/30. Analysis by SDS-PAGE showed a unique polypeptide band with Coomassie Blue stain and nondenaturing PAGE of the purified enzyme obtained from media with different carbon sources showed the band, too, when stained for glucose oxidase activity. The optimal hydrolysis temperature for sucrose, raffinose and inulin was 55°C and the optimal pH for sucrose was 4.75. The apparent K _m values for sucrose, raffinose and inulin are 4.58, 7.41 and 86.9 mg/ml, respectively. Thin layer chromatography showed that inulinase from K. marxianus var. bulgaricus was capable of hydrolyzing different substrates (sucrose, raffinose and inulin), releasing monosaccharides and oligosaccharides. The results obtained suggest the hypothesis that enzyme production was constitutive. Journal of Industrial Microbiology & Biotechnology (2000) 25, 63–69. Received 17 November 1999/ Accepted in revised form 30 May 2000     

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.     

Properties of rat liver plasma membrane adenylate cyclase after chromatography on O-diethylaminoethyl-cellulose and agarose-hexane-GTP: Sensitivity of partially purified and purified enzyme to Lubrol-PX, 5′-guanylylimidodiphosphate,and glucagon

Partially purified rat liver plasma membranes were enriched to yield a more glucagon-sensitive membrane fraction which was solubilized with Lubrol-PX. The supernate obtained after centrifugation at 165,000g was subjected to O-diethylaminoethyl anion exchange chromatography. An adenylate cyclase fraction was eluted and purified further by chromatography on agarose-hexane-GTP. The enzyme adsorbed to the affinity resin and was eluted with 0.5 m Tris-HCl. The protein isolated by chromatography on the affinity resin was homogenous by conventional acrylamide gel electrophoresis; one band was observed in sodium dodecyl sulfate. The purified enzyme was free of nucleotide phosphohydrolases found in the parent solubilized membrane preparation. The anion exchange product was not sensitive to glucagon; Lubrol-PX and 5′-guanylylimidodiphosphate [Gpp(NH)p] decreased the activity of this fraction. In the presence of detergent or guanyl nucleotide, glucagon, at 10^?6m, increased enzyme activity by 30 and 21%, respectively, to a statistically significant degree, but not above basal levels. Adenylate cyclase was also purified by subjecting the 165,000g supernate directly to agarose-hexane-GTP; agarose-hexane-ATP or agarose-hexane was not effective. The affinity-derived material was associated with 85 nmol of Lubrol-PX/mg of protein. When calculated on the basis of a molecular weight of 150,000 for detergent-free protein after gel filtration on Bio-Gel A-0.5 m, there was 13 mol of detergent/mol of the enzyme obtained by chromatography on the affinity resin. The direct affinity product was insensitive to glucagon and Gpp(NH)p; enzyme activity varied as a function of Lubrol concentration.     

Purification and Some Properties of α-Galactosidase from Tubers of Stachys affinis

An α-galactosidase from tubers of S. affinis was purified about 130 fold by ammonium sulfate fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-75. The purified enzyme showed a single protein band on disc gel electrophoresis. The molecular weight of the enzyme was determined to be approximately 42,000 by gel filtration and 44,000 by SDS disc gel electrophoresis. The optimum reaction pH was 5.2. The enzyme hydrolyzed raffinose more rapidly than planteose. The activation energy of raffinose and planteose by the enzyme was estimated to be 7.89 and 11.4 kcal/mol, respectively. The enzyme activity was inhibited by various galactosides and structural analogs of d-galactose. Besides hydrolytic activity, the enzyme also catalyzed the transfer reaction of d-galactosyl residue from raffinose to methanol.     

Purification of Rabbit Liver Guanine Aminohydroiase

Abstract

Rabbit liver guanine aminohydrolase has been purified 1250-fold by utilization of an affinity chromatographic separation on 9-(p-aminoethoxyphenyl)guanine-Sepharose with 50% recovery of activity. Polyacrylamide gel electrophoresis of the purified preparations revealed several protein bands which corresponded to regions of enzyme activity measured on gels which had been run under the same conditions. Gel concentration studies of the protein migration rate showed that the protein bands differed in molecular size. The minimum molecular weight was 100, 000 from gel permeation chromatography studies. The pH optimum was near pH 8 and the Km, with guanine as substrate was 5.6 × 10^?6M. The latter values are in close agreement with partially purified preparations described in the literature.     

Purification and partial characterization of an acid phosphatase from the body wall of sea cucumber Stichopus japonicus

A high molecular weight (HMW) acid phosphatase from the body wall of sea cucumber Stichopus japonicus was purified to homogeneity by a combination of anion exchange chromatography, gel filtration chromatography and high performance liquid chromatography (HPLC). The enzyme was purified 19.3-fold with a total yield of 1.2%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of MW 147.9 kDa. The enzyme displayed maximum activity at pH 4.0 and 50 °C with p-nitrophenyl phosphate as substrate. The enzyme activity appeared to be stable over pH 2.0–5.0 and up to 40 °C. The enzyme activity was enhanced slightly by Mg²⁺, whereas inhibited strongly by Cu²⁺ and Zn²⁺. The enzyme hydrolyzes several phosphate esters, suggesting a probable non-specific nature. The amino acid sequences of three segments of the purified enzyme were analyzed by mass spectroscopy, which did not have any homology with previously described acid phosphatase.     

Partial purification and characterization of platelet factors stimulating the multiplication of normal human glial cells

Multiplication-stimulating activity for human glial cells was purified from human outdated platelets. By ion exchange chromatography anionic activity was separated from cationic activity. The former could be further separated by Sephadex G-200 gel chromatography into two peaks, whose molecular weights were 40 000 and < 10 000. The cationic activity was partially purified by concanavalin A (ConA) Sepharose chromatography, hydroxylapatite chromatography and SDS-polyacrylamide gel electrophoresis. The cationic activity was heterogeneous as demonstrated by isoelectric focusing (Ip 9.5–10.4), gel filtration on Bio-Gel P-150 and SDS-polyacrylamide gel electrophoresis (mol. wt 26 000–33 000). Less than 50 ng/ml was required of the factor to give a glial cell stimulation corresponding to that afforded by 1 % of human serum. A thymidine-degrading enzyme, present in human platelets and to a low degree also in human serum, was found to interfere with the assay for multiplication-stimulating activity. The enzyme (probably a thymidine phosphorylase) converted [³H]thymidine to [³H]thymine, causing a reduced incorporation of ³H into cellular DNA. This difficulty was circumvented by use of an autoradiographic estimation (per cent labelled nuclei) of the multiplication-stimulating activity.     

A New Peptide-N 4-(acetyl-β-glucosaminyl)asparagine Amidase from Soybean (Glycine max) Seeds: Purification and Substrate Specificity

We report here the isolation and characterization of a peptide-N ⁴-(acetyl-β-glucosaminyl) asparagine amidase (peptide: N-glycanase) from soybean (Glycine max) seeds. The enzyme was purified to homogeneity with 6.5% yield from defatted soybean meal extract by ion-exchange chromatography, gel filtration, hydroxyapatite chromatography, and hydrophobic chromatography. The purified enzyme, designated PNGase-GM, had the apparent molecular mass of 93 kDa by SDS-PAGE and 90 kDa by gel filtration, indicating this PNGase is a monomeric protein. The enzyme showed maximal activity at pH 4.5-5.0. PNGase-GM was capable of hydrolyzing the β-aspartylglycosylamine linkage (GlcNAcβ1→Asn) of various glycopeptide substrates bearing high-mannose type, hybrid type, and xylose/fucose-containing plant complex type N-glycan units, while this amidase was far less active on the glycopeptides bearing sialylated animal complex-type glycans.     

Purification and properties of milk-clotting enzyme from Bacillus subtilis K-26

A milk-clotting enzyme from Bacillus subtilis K-26 was purified by gel filtration and ion-exchange chromatography resulting in a 24-fold increase in specific activity with an 80% yield. Polyacrylamide gel electrophoresis and ultracentrifugel analysis revealed that the purified enzyme was homogeneous and had a molecular weight of 27,000 and a K_m of 2.77mg/ml for κ-casein. The enzyme was most stable at pH 7.5 and showed increasing clotting activity with decrease in milk pH up to 5.0. The maximum milk-clotting activity was obtained at 60°C, but the enzyme was inactivated by heating for 30 min at 60°C. The enzyme was irreversibly inhibited by EDTA and unaffected by DFP. Heavy-metal ions (Hg²⁺, Pb²⁺) inactivated the enzyme.     

Characterization and purification of thermostable beta-glucosidase from Clostridium thermocellum.

A β-glucosidase was isolated from $\text{Clostridium thermocellum}$; the enzyme was localized in the periplasmic space.It was purified in a five-step procedure including ion-exchange chromatography on DEAE-Cellulose, chromatography on HA-Ultrogel and DEAE-Sephadex, gel filtration on AcA 34 Ultrogel and isoelectric focusing.The final preparation was purified 944-fold with a recovery of about 5% of the initial enzyme activity.Polyacrylamide disc electrophoresis of the purified enzyme gave a single band at pH 8.3. The enzyme is active towards cellobiose and p-nitrophenyl-β-D-glucoside(PNPG) and developed maximum activities at pH 6.0 and 65°C. A molecular weight of 50,000 daltons was estimated by gel filtration and the enzyme was isoelectric at pH 4.68.     

A rapid method for the purification of S-adenosylmethionine: Protein-carboxyl O-methyltransferase by affinity chromatography

A simple method to purify S-adenosylmethionine: protein-carboxyl O-methyltransferase (protein methylase II, EC 2.1.1.24) from calf brain has been developed using affinity chromatography. The product of the reaction, S-adenosyl-l-homocysteine, which is a competitive inhibitor of the enzyme, was covalently linked to Sepharose beads. This gel proved to be an effective binder for protein methylase II at pH 6.2 and allowed for specific removal of the enzyme by the addition of the methyl donor substrate, S-adenosyl-l-methionine to the elution buffer. One step using this affinity chromatography column resulted in 377-fold purification of the enzyme and 71% recovery of the activity. Subsequent Sephadex G-100 chromatography enabled the enzyme to be purified 3000-fold from the calf brain whole homogenate. The purified enzyme showed a number of protein methylase II activity peaks following preparative gel electrophoresis with one major enzyme peak.     

Optimization and purification of glucansucrase produced by Leuconostoc mesenteroides DRP2-19 isolated from Chinese Sauerkraut

Abstract

Strain DRP2-19 was detected to produce high yield of glucansucrase in MRS broth, which was identified to be Leuconostoc mesenteroides. In order for industrial glucansucrase production of L. mesenteroides DRP2-19, a one-factor test was conducted, then response surface method was applied to optimize its yield and discover the best production condition. Based on Plackett–Burman (PB) experiment, sucrose, Ca²⁺, and initial pH were found to be the most significant factors for glucansucrase production. Afterwards, effects of the three main factors on glucansucrase activity were further investigated by central composite design and the optimum composition was sucrose 35.87?g/L, Ca²⁺ 0.21?mmol/L, and initial pH 5.56. Optimum results showed that glucansucrase activity was increased to 3.94?±?0.43?U/mL in 24?hr fermentation, 2.66-fold higher than before. In addition, the crude enzyme was purified using ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of glucansucrase was determined as approximately 170?kDa by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified 15.77-fold and showed a final specific activity of 338.56?U/mg protein.     

Purification and Some Properties of Chlorogenic Acid Oxidase from Apple (Malus pumila)

Chlorogenic acid oxidase was extensively purified to homogeneity from apple flesh (Malus pumila cv. Fuji). The enzyme was purified 470-fold, with a total yield close to 70% from the plastid fraction by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography. The molecular weight was determined to be 65,000 by both SDS-PAGE and gel filtration chromatography. The optimum pH for the enzyme activity was around 4.0, and the enzyme was stable in the range of pH 6–8. The pI obtained by isoelectrofocusing was 5.4, and the N-terminal amino acid sequence was N-Asp-Pro-Leu-Ala-Pro-Pro-. The reaction rate of the purified enzyme was much larger for chlorogenic acid than for other o-diphenols such as (+)-catechin, (?)-epicatechin and 4-methylcatechol, and the enzyme lacked both cresolase activity and p-diphenol oxidase activity. The K_m value for the enzyme was found to be 122μM toward chlorogenic acid. The purified enzyme had far less thermal stability than the enzyme of the plastid fraction. Diethyl-dithiocarbamate, sodium azide, o-phenanthroline and sodium fluoride markedly inhibited the enzyme activity.     

Studies on the Lipase of Thermophilic Fungus Humicola lanuginosa

A protease occurring in the endosperm fraction of germinating corn was purified by means of (NH₄)₂SO₄ fractionation, CM-celluIose chromatography, DEAE-cellulose chromatography, Sephadex G-100 gel filtration and preparative polyacrylamide gel electrophoresis. The purified protease was found to have a molecular weight of about 21,000 and an isoelectric point of pH 2.3 or lower. The optimum pH was found to lie at 3.0 when measured with denatured hemoglobin as substrate. The protease was generally activated by thiol compounds and completely inhibited by p-chloromercuribenzoic acid. Neither diisopropylphosphofluoridate nor diazoacetyl-dl-norleucine methyl ester affected the protease activity. Antipain greatly inhibited the protease action whereas pepstatin had no significant effect. These data indicate, in conclusion, that the protease possesses a unique property to be a sulfhydryl enzyme most active in an acidic region around pH 3.