Selection of High Ubiquinone 10-Producing Strain of Tobacco Cultured Cells by Cell Cloning Technique

A novel enzyme, which was named N^α-benzyloxycarbonyl amino acid urethane hydrolase, was purified from a cell-free extract of Streptococcus faecalis R ATCC 8043, using N^α-benzyloxycarbonyl glycine as substrate. The enzyme was purified 1300-fold with an activity yield of 8%. The purified enzyme was homogeneous by disc electrophoresis. The molecular weight of the native enzyme is about 220,000 by gel filtration, and a molecular weight of 32,000 was determined for the reduced and denatured enzyme by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point was 4.48. The enzyme was inhibited by p-chloromercuribenzoate. The presence of divalent cations (i.e., Co²⁺ or Zn²⁺) is essential for its activity.     

Purification and Properties of Extracellular Acid Phosphatase from Zizania latifolia-pavasite,stilago esculenta

An extracellular acid phosphatase from Ustilago esculenta was purified to homogeneity on the basis of polyacrylamide gel electrophoresis. It was a glycoprotein with an isoelectric point of 4.7. The molecular weight of the enzyme was estimated to be about 343,000 by gel filtration on Sephadex G-200, whereas on SDS-polyacrylamide gel electrophoresis, the enzyme gave a single protein band with a molecular weight of 116,000. This result suggests that the enzyme consists of three identical subunits. The enzyme showed an optimum activity at pH 4.5, retained 90% of its activity for 10 min at 55°C and had a Km value of 0.25 mm for p-nitrophenylphosphate. No definite substrate specificity of the enzyme was observed.     

The Isolation and Physico-chemical Properties of Crystalline Quinolinate Phosphoribosyltransferase from Hog Liver

Quinolinate phosphoribosyltransferase (an intermediary enzyme in the de novo NAD biosynthetic pathway) was purified from hog liver and its crystallization from a mammal was successful for the first time. This crystalline enzyme preparation was certified to be homogeneous by ultracentrifugal analysis and polyacrylamide gel disc electrophoresis. Its molecular weight was 173,000 using the gel filtration method, and 172,000 using sedimentation velocity analysis. The subunit molecular weight was estimated at 33,500 with SDS polyacrylamide gel disc electrophoresis. Several physico-chemical parameters were also determined.     

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.     

Isolation and Characterization of Nα-Benzyloxycarbonyl Amino Acid Urethane Hydrolase II from Lactobacillus fermenti 36 ATCC 9338

A novel enzyme, which was named N^α-benzyloxycarbonyl amino acid urethane hydrolase II, was purified from a cell-free extract of Lactobacillus fermenti 36 ATCC 9338. The enzyme catalyzed the stoichiometric hydrolysis of N^α-benzyloxycarbonyl arginine to form benzyl alcohol and arginine. The enzyme was purified 106-fold with an activity yield of 3%. The purified enzyme was homogeneous by disc gel electrophoresis. The molecular weight of the native enzyme is about 200,000 by gel filtration, and a molecular weight of 27,000 was found for the reduced and denaturated enzyme by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point of the enzyme was 5.0, it was inhibited by disodium ethylenediamine tetraacetate and p-chloromercuribenzoate, and the presence of a divalent cation, i.e. Co²⁺, is essential for its activity.     

Glycogen Formation from Glycols and their Esters in the Livers of Chicks

Purification was conducted on polyvinyl alcohol (PVA) degrading enzyme produced and secreted into the culture medium by Pseudomonas O–3 strain. The enzyme was found to separate into several fractions by ion-exchange chromatography and gel filtration. Among these fractions, a fraction adsorbed to SP-Sephadex C–50 at pH 6.0 was purified to homogeneity by polyacrylamide gel electrophoresis. Some properties of this purified enzyme were examined. Optimum pH and temperature were 9.0 and 40°C, respectively. The enzyme was stable up to 50°C and in a pH range between 5 and 11 at 5°C. The enzyme activity was inhibited by Co²⁺, Ni²⁺, EDTA, hydroxylamine and salicylaldoxime. In substrate specificity, this enzyme oxidized several kinds of modified PVA, as well as normal PVA, but did not oxidize other synthetic polymers, such as vinylon, polyacrylamide and polyvinyl acetate. The effect of oxygen on this enzyme was examined, and without oxygen, PVA was not broken down by this enzyme. The molecular weight of this enzyme was estimated by gel filtration on Sephadex G–100 to be approximately 26,000.     

Studies on the characterization of the sodium-potassium transport adenosine triphosphatase. Purification and properties of the enzyme from the electric organ of Electrophorus electricus

An unspecific carboxylesterase was purified 180-fold from acid-precipitated human liver microsomes. The final preparation was homogeneous on disc electrophoresis and polyacrylamide gel electrophoresis in the presence of 6.25 M urea at pH 3.2. A single symmetrical peak was also found on gel filtration and on velocity sedimentation in the analytical ultracentrifuge, whereas slight heterogeneity was observed on isoelectric focusing.The amino acid composition of the purified enzyme is presented. From the results the partial specific volume (0.745 ml × g^?1) and the minimal molecular weight (60,000) could be calculated. Fingerprint maps of tryptic peptides from the carboxymethylated enzyme are shown.The molecular weight as determined by gel filtration, disc electrophoresis, and analytical ultracentrifugation is in the range of 181,000–186,000. For the molecular weight of the subunits a value of 61,500 has been obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The equivalent weight of the enzyme has been estimated to be 62,500 from stoichiometry of its reaction with diethyl-p-nitrophenyl-phosphate. Partial cross-linking of the subunits with dimethyl suberimidate and subsequent sodium dodecylsulfate polyacrylamide gel electrophoresis yielded three bands with molecular weights of 60,000, 120,000, and 180,000.From these results it is concluded that human liver esterase is a trimeric protein. It is composed of three subunits of equal size, and there is one active site per subunit.     

trans-2, cis-4-Heptadienoic Acid,One of Metabolites of Sporobolomyces odorus

Water-soluble phospholipase B was purified to homogeneity from Torulaspora delbrueckii cell washings. The washings were concentrated by ultrafiltration, and then a fraction with phospholipase B activity was precipitated with ammonium sulfate, and purified by sequential column chromatographies on Octyl-Sepharose CL-4B, DEAE-Sephacel, and Sepharose 6B. The molecular weight of the enzyme was estimated to be 170,000~200,000 by SDS-polyacrylamide gel electrophoresis and by gel filtration with a Sephadex G-200 column. The isoelectric point of the enzyme was 4.0. The purified enzyme had two pH optima at pH 2.5 and pH 7.5. The activity at acidic pH was greatly stimulated by the divalent metal ions tested, but the activity at alkaline pH was stimulated mainly by Ca²⁺ and Fe²⁺. The purified enzyme had both lysophospholipase activity and phospholipase B activity in a ratio of 37:1 at acidic pH and 73:1 at alkaline pH. The amino acid composition of the enzyme was characterized by high contents of Asp, Ser, Leu, and Gly.     

Purification and Properties of a Phenylcarbamate Herbicide Degrading Enzyme of Pseudomonas alcaligenes Isolated from Soil

A phenylcarbamate degrading enzyme was isolated from Pseudomonas alcaligenes. The enzyme was purified to a specific activity of 119U/mg by ammonium sulphate precipitation, gel filtration, DEAE and hydroxy-apatite chromatographies. The purified enzyme was found to be homogeneous on SDS polyacrylamide gel electrophoresis. The molecular weight was estimated to 68,000. The pH optimum was around 9.5 and the temperature optimum was 28°C. The Km for CIPC was 1.19 × 10^–5m. Hg^{2 +}, PMSF inhibited the enzyme, but thiol reagents and EDTA had no effect. The enzyme degraded a number of phenylcarbamate herbicides (CIPC, BIPC, IPC and swep) and propanil but did not hydrolyse bar ban and carbetamide, which are phenylcarbamates, or monuron and linuron, which are phenylureas. The enzyme is probably an amidase.     

Multiple Shoot Formation from Almond Seeds and an Excised Single Shoot

An amylase which produces maltotriose from starch as the main product was found in the culture filtrate of a strain of Bacillus subtilis newly isolated from soil. The enzyme was purified to almost complete homogeneity, as judged by disc electrophoresis in polyacrylamide gel, by means of ammonium sulfate fractionation, DEAE-Sepharose column chromatography and Sephadex gel filtration.

The optimum pH and temperature of the enzyme were around 6~7 and 50°C, respectively. Metal ions such as Hg²⁺, Cu²⁺, Zn²⁺, Ni²⁺ and Fe²⁺ strongly inhibitied the enzyme activity. The molecular weight was found to be about 25,000 by gel filtration. The yields of maltotriose from short-chain amylose (DP 17), amylopectin, soluble starch and glycogen were about 69, 56, 56 and 40%, respectively.     

Purification and Some Properties of Carboxylesterase from Arthrobacter globiformis; Stereoselective Hydrolysis of Ethyl Chrysanthemate

An esterase with excellent stereoselectivity for (+)-trans-ethyl chrysanthemate was purified to homogeneity from Arthrobacter globiformis SC-6-98-28. The purified enzyme hydrolyzed a mixture of ethyl chrysanthemate isomers stereoselectively to produce (+)-trans-acid with 100% stereoisomeric purity. The apparent molecular weight of the purified enzyme was 43,000 on SDS–polyacrylamide gel electrophoresis, and 94,000 on gel filtration chromatography. The optimum conditions for the ester hydrolysis were pH 10.0 at 45°C. The purified esterase hydrolyzed short-chain fatty acid esters, but did not have detectable activity on long-chain water-insoluble fatty acid esters. The enzyme activity was inbibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride.     

Purification and Properties of Pyranose Oxidase from Coriolus versicolor

Coriolus versicolor KY2912 grown on a medium containing glucose, sucrose or glycerol produced pyranose oxidase. Pyranose oxidase (glucose-2-oxidase) was purified by HPA-75 chromatography, Sepharose 4B and Sephadex G-100 gel filtration, and hydroxyapatite chromatography. The purified enzyme preparation showed a single protein band on acrylamide gel electrophoresis. The highest activity was obtained when D-glucose was employed as substrate and molecular oxygen as electron acceptor. The enzyme was most active at pH 6.2 and 50°C, stable in the pH region between 5.0 and 7.4, and the activity was completely lost above 70°C. The activity was inhibited by Ag⁺ , Cu²⁺ and PCMB. The enzyme contained FAD covalently bound to the polypeptide chain. The enzyme consisted of identical subunits with a molecular weight of 68,000, and showed a total molecular weight of 220,000.     

Purification and Characterization of the Acidic Lectin from Winged Bean Seeds

Winged bean acidic lectin was purified by DEAE-Sephadex A-50 and affinity chromatography on N-acetylgalactosamine-agarose gel. The purified lectin was a glycoprotein homogeneous on polyacrylamide gel electrophoresis, isoelectric focusing, and gel filtration. The molecular weight of the lectin was 52,000 by gel filtration, and SDS-polyacrylamide gel electrophoresis gave a single component of molecular weight of 27,000. Its isoelectric point was 5.5. The acidic lectin was rich in acidic amino acids, and contained 2mol of methionine but no cystine. It also agglutinated both trypsinized and untreated human erythrocytes (types A, B, AB and O), but not rabbit erythrocytes. The hemagglutination was inhibited by d-galactose and related sugars. Modification of the acidic lectin with N-bromosuccinimide caused a concomitant loss of the hemagglutinating activity with oxidation of tryptophan residue. The acidic lectin was immunologically different from the purified winged bean basic lectin by double immunodiffusion using antiserum raised against the basic lectin.     

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 Glucoamylase from Lactobacillus amylovorus ATCC 33621

An intracellular glucoamylase (E.C. 3.2.1.3) was purified to homogeneity from Lactobacillus amylovorus on a Fast Protein Liquid Chromatography System (FPLC) with a Mono Q ion-exchanger and two Superose 12 gel filtration columns arranged in series. The enzyme activity was quantified with a specific, chromogenic substrate, p-nitrophenyl-β-maltoside. Preparative gel electrophoresis was then used to further purify active enzyme fractions. Native polyacrylamide gel electrophoresis (Native-PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of molecular weight 47 kDa. Glucoamylase activity of the purified protein was confirmed by its ability to degrade starch on a 0.025% starch-polyacrylamide gel stained with I₂/KI. Glucoamylase exhibited optimum catalytic activity at pH 6.0 and 45°C, and the enzyme had an isoelectric point near 4.39. The glucoamylase contained high levels of hydrophilic amino acids, comparable to fungal glucoamylases. Received: 12 July 1996 / Accepted: 10 September 1996     

Isolation and characterisation of cathepsin-B from bovine pancreas

A simple procedure for the isolation of cathepsin-B from bovine pancreas employing ammonium sulphate fractionation, DEAE cellulose chromatography and Sephadex G-200 gel filtration is described. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The molecular weight as determined by gel filtration of the enzyme was 26,850. ItsK _m andV _max values were 12.8 mM and 0.303 Μmol/min/mg, respectively. TheK _i for iodoacetamide was 0.16 mM.     

Purification and Properties of a Lytic Enzyme from Streptomyces griseus H-402

A lytic enzyme which was capable of lysing cells of Streptococcus mutans was purified from the culture filtrate of Streplomyces griseus H–402 by Amberlite CG–50 treatment, CM-cellulose and hydroxylapatite column chromatographies, and Sephadex G–150 gelfiltration. The lytic enzyme was obtained in a crystalline form which was homogeneous in polyacrylamide gel electrophoresis. The molecular weight was estimated to be 2×10⁴ by the thin-layer gel-filtration method on Sephadex G–75, and 2.3 × 10⁴ by the method of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was found to be a N-acetylmuramidase whose activity was lost by N-bromosuccinimide as an inhibitor.     

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 characterisation of leukotriene A4 hydrolase from rat neutrophils

Leukotriene A₄ hydrolase was rapidly and extensively purified from rat neutrophils using anion exchange and gel filtration high-pressure liquid chromatography. The enzyme which converts the allylic epoxide leukotriene A₄ to the 5,12-dihydroxyeicosatetraenoic acid leukotriene B₄ was localized in the cytosolic fraction and exhibited an optimum activity at pH 7.8 and apparent K_m for leukotriene A₄ between 2 · 10^?5 and 3 · 10^?5 M. The purified leukotriene A₄ hydrolase was shown to have a molecular weight of 68 000 on sodium dodecylsulfate polyacrylamide gel electrophoresis and of 50 000 by gel filtration. The molecular weight and monomeric native form of this enzyme are unique characteristics which distinguish leukotriene A₄ hydrolase from previously purified epoxide hydrolases.     

PURIFICATION OF L-GLUTAMIC ACID DECARBOXYLASE FROM CATFISH BRAIN

—L-Glutamic acid decarboxylase (GAD) from brain of the channel catfish (Ictalurus punctatus) has been purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, calcium phosphate gel and preparative polyacrylamide gel electrophoresis. The purity of the enzyme preparation was established by showing that on both 7.5% regular and 3.7–15% gradient polyacrylamide gel electrophoresis the enzyme migrated as a single protein band which contained all the enzyme activity. The molecular weight of the purified GAD was estimated by gel filtration and gradient polyacrylamide gel to be 84,000 ± 2000 and 90,000 ± 4000, respectively. SDS-polyacrylamide gel electrophoresis revealed three major proteins with molecular weights of 22,000 ± 2000, 40,000 ± 5000 and 90, 000 ± 6000 which may represent a monomer, dimer, and tetramer. Antibodies against the purified enzyme were obtained from rabbit after four biweekly injections with a total of 80 μg of the enzyme. A double immunodiffusion test using these antibodies and a crude extract from catfish brains showed only a single, sharp precipitin band which still retained the enzyme activity, suggesting that the precipitin band was indeed a GAD-anti-GAD complex. In an enzyme inhibition study, a maximum inhibition of 60–70% was obtained at a ratio of GAD protein/anti-GAD serum of about 1:1.6. Furthermore, the precipitate from the GAD-anti-GAD incubation mixture also contained the enzyme activity, suggesting that the antibody was specific to GAD and that the antigen used was homogeneous. Advantages and drawbacks of the purification procedures described here and those used for mouse brain preparations are also discussed.