Purification and properties of bromoperoxidase from Pseudomonas pyrrocinia

A bromoperoxidase was purified and partially characterized from Pseudomonas pyrrocinia ATCC 15958, a bacterium that produces the antifungal antibiotic pyrrolnitrin. The purified enzyme preparation was homogeneous as determined by polyacrylamide gel electrophoresis and ultracentrifugation. The molecular mass of the enzyme was estimated to be 154 kDa +/- 3 kDa as determined by gel filtration and ultracentrifugation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a single band with the mobility of a 76-kDa species. Therefore, in solution at neutral pH, bromoperoxidase exists as a dimeric species. The isoelectric point was 5.0. The prosthetic group of this procaryotic bromoperoxidase was ferriprotoporphyrin IX. The spectral properties of the native and reduced enzyme are reported. The purified enzyme showed brominating as well as peroxidase and catalase activity.     

Purification and molecular and catalytic properties of bromoperoxidase from Streptomyces phaeochromogenes

A bromoperoxidase has been isolated and purified from the chloramphenicol-producing actinomycete Streptomyces phaeochromogenes. The purified enzyme was homogeneous as determined by polyacrylamide gel electrophoresis. The prosthetic group of the bromoperoxidase was ferriprotoporphyrin IX. Based on gel filtration results the molecular weight of the enzyme was 147 000 +/- 3000. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed a single band having the mobility of a 72 500 molecular weight species. Therefore, in solution at neutral pH, the bromoperoxidase behaved as a dimer. The isoelectric point was 4.0. The spectral properties of the native and reduced enzyme are reported. The homogeneous enzyme also had peroxidase and catalase activity.     

Purification and characterization of a novel bacterial non-heme chloroperoxidase from Pseudomonas pyrrocinia

The first bacterial chloroperoxidase that is capable of catalyzing the chlorination of indole to 7-chloroindole was detected in Pseudomonas pyrrocinia ATCC 15958, a bacterium that produces the antifungal antibiotic pyrrolnitrin (Wiesner, W., van Pée, K.H., and Lingens, F. (1986) FEBS Lett. 209, 321-324). Here we describe the purification and characterization of this bacterial non-heme chloroperoxidase. The enzyme was purified by DEAE-cellulose chromatography at different pH values, molecular sieve chromatography, and Bio-Gel HTP hydroxylapatite. After the last purification step, chloroperoxidase was homogeneous by polyacrylamide gel electrophoresis and ultracentrifugation. Based on gel filtration and ultracentrifugation results, the molecular weight of the enzyme was 64,000 +/- 3,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band with the mobility of a 32,000 molecular weight species. Therefore, in solution at neutral pH, this chloroperoxidase is a dimer. The enzyme did not exhibit any absorbance in the visible region of the spectrum. The isoelectric point was 4.1. Chloroperoxidase was specific for I-, Br-, and Cl- and was not inhibited by azide, but was inhibited by cyanide and F-. This procaryotic chloroperoxidase catalyzed the bromination of monochlorodimedone but not its chlorination and has no peroxidase or catalase activity. The pH optimum of the enzyme was between 4.0 and 4.5, and the enzyme was stable between pH 3.5 and 8.5 and showed no loss of activity when incubated at 60 degrees C for 2 h. Chloroperoxidase also chlorinated 4-(2-amino-3-chlorophenyl) pyrrole to yield aminopyrrolnitrin, the immediate precursor of pyrrolnitrin. This suggests very strongly that chloroperoxidase is involved in the biosynthesis of the antibiotic pyrrolnitrin.     

Purification of bromoperoxidase from Corallina pilulifera

Bromoperoxidase was purified from the crude extract of Corallina pilulifera (Corallinaeae, Rhodophyta) and found to be homogeneous upon disc gel electrophoresis by precipitation of ammonium sulfate and sequential column chromatographies of DEAE-Sepharose CL-6B, Sepharose 6B and Cellulofine GC-700m. The purified enzyme did not exhibit optical absorption spectra of a hemoprotein. Therefore, bromoperoxidase of C. pilulifera was completely distinguishable from other haloperoxidases which have heme-irons at the catalytic sites.     

Purification and characterization of ovine pancreatic alpha - amylase.

Ovine pancreatic amylase has been purified from pancreas homogenate by ammonium sulfate, acetone precipitation, DEAE-cellulose chromatography and finally by specific adsorption on polydextran gel. The enzyme is homogeneous and found as a single form as shown by disc electrophoresis, SDS gel electrophoresis, electrofocusing and ultracentrifugation. Its specific activity is similar to that of porcine amylase. The amino acid composition indicates a high content in aromatic and acidic amino acids as for the porcine enzyme; however the methionine and half cystine content differ widely. The N-terminal end is blocked. Also ovine amylase is glycosylated. The molecular weight (56,000-58,000) is slightly higher than for the porcine enzyme. The isoelectric point is acidic (pI = 3.2).     

龙须菜中溴过氧化物酶的分离纯化及酶学性质分析

对中国北方海域江蓠属养殖龙须菜(Gracilaria lemaneiformis)进行了溴过氧化物酶分离纯化及性质的研究。粗提液中酶催化检测反应不稳定, 活力单位较低或无; 经DEAE cellulose 52离子交换层析, 去除了结构多糖及藻胆蛋白, 酶催化反应稳定, 得到比活力为2.8的电泳纯溴过氧化物酶。对纯化溴过氧化物酶性质研究表明: 该溴过氧化物酶为单体酶, 分子量约66 kD, 溴化单氯双甲酮时的最适pH值为6.0, 在40°C以下和pH 3.0~9.0之间有很好的稳定性。钒酸盐可提高该溴过氧化物酶的催化活性, 而Fe2+、Fe3+、Cu2+、Zn2+和EDTA等化合物对其有较显著的抑制作用。反应动力学实验表明, 该酶对Br-、H2O2的Km分别为53.5 mmol/L和38 mmol/L。     

Purification and physico-chemical properties of glutamine synthetase from pea chloroplasts]

A highly purified, practically homogeneous glutamine synthetase was isolated from pea leaf chloroplasts. The enzyme purity was assayed by polyacrylamide gel electrophoresis and analytical ultracentrifugation. The sedimentation coefficient is 16,3S. The sedimentation equilibrium analysis showed that the molecular weight of the enzyme is equal to 480 000. The minimal molecular weights of the enzyme as calculated from the data of polyacrylamide gel electrophoresis in the presence of SDS and the amino acid analysis were found to be 62 000 and 60 000, respectively. The enzyme contains a large amount of dicarboxylic and sulfur-containing amino-acids. The N-terminal amino acid is glycine. The isoelectric point for the enzyme lies within the pH range of 4,2-4-4.     

龙须菜中溴过氧化物酶的分离纯化及酶学性质分析

对中国北方海域江蓠属养殖龙须菜(Gracilaria lemaneiformis)进行了溴过氧化物酶分离纯化及性质的研究。粗提液中酶催化检测反应不稳定, 活力单位较低或无; 经DEAE cellulose 52离子交换层析, 去除了结构多糖及藻胆蛋白, 酶催化反应稳定, 得到比活力为2.8的电泳纯溴过氧化物酶。对纯化溴过氧化物酶性质研究表明: 该溴过氧化物酶为单体酶, 分子量约66 kD, 溴化单氯双甲酮时的最适pH值为6.0, 在40°C以下和pH 3.0~9.0之间有很好的稳定性。钒酸盐可提高该溴过氧化物酶的催化活性, 而Fe2+、Fe3+、Cu2+、Zn2+和EDTA等化合物对其有较显著的抑制作用。反应动力学实验表明, 该酶对Br-、H2O2的Km分别为53.5 mmol/L和38 mmol/L。     

Detection of a new chloroperoxidase in Pseudomonas pyrrocinia

A new chloroperoxidase could be detected in Pseudomonas pyrrocinia ATCC 15,958, a bacterium that produces the antifungal antibiotic pyrrolnitrin. This enzyme was separated from a ferriprotoporphyrin IX containing bromoperoxidase which was also produced by this bacterium. The enzyme is capable of catalyzing the chorination of indole to 7-chloroindole. This procaryotic chloroperoxidase requires the presence of H2O2 and can also brominate monochlorodimedone, but cannot catalyze its chlorination. This enzyme is the first chloroperoxidase described from procaryotic sources.     

1, 4-alpha-Glucan phosphorylase from Klebsiella pneumoniae purification, subunit structure and amino acid composition.

1. A 1,4-alpha-glucan phosphorylase from Klebsiella pneumoniae has been purified about 80-fold with an over-all yield greater than 35%. The purified enzyme has been shown to be homogeneous by gel electrophoresis at different pH-values, by isoelectric focusing, by dodecylsulfate electrophoresis and by ultracentrifugation. 2. The molecular weight of the native enzyme has been determined to be 180 000 by ultra-centrifugation studies, in good agreement with the value of 189 000 estimated by gel permeation chromatography. 3. The enzyme dissociates in the presence of 0.1% dodecylsulfate or 5 M guanidine hydrochloride into polypeptide chains. The molecular weight of these polypeptide chains has been found to be 88 000 by dodecylsulfate polyacrylamide gel electrophoresis and 99 000 by sedimentation equilibrium studies, indicating that the native enzyme is composed of two polypeptide chains. 4. The enzyme contains pyridoxalphosphate with a stoichiometry of two moles per 180 000 g protein, confirming that the 1,4-alpha-glucan phosphorylase from Klebsiella pneumoniae is a dimeric enzyme. 5. The amino acid composition of the enzyme has been determined, and its correspondence to that of 1,4-alpha-glucan phosphorylases from other sources is discussed. 6. The pI of the enzyme has been shown to be 5.3 and its pH-optimum to be about pH 5.9. The enzyme is stable in the range from pH 5.9 to 10.5.     

Purification and Properties of Endo-polygalacturonase from Aspergillus japonicus

An endo-polygalacturonase from culture extracts of Aspergillus japonicus was purified about 34-fold by ammonium sulfate fractionation, SE-Sephadex column chromatography and gel filtration. The purified enzyme was homogeneous on ultracentrifugation and disc electrophoresis. Using gel filtration a molecular weight of 35,500 was estimated for the enzyme. The enzyme rapidly reduced the viscosity of pectic acid and released reducing groups in a random manner, yielding a mixture of mono-, di- and trigalacturonic acids as end products. The pH optimum of the enzyme for viscosity-reducing activity was 4.5 with pectin and pectic acid as substrates, and that for releasing reducing groups was also 4.5 with various pectic substances. The purified enzyme was able to macerate various kinds of plant tissues by itself.     

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.     

Isolation and characterization of argininosuccinate synthetase from human liver

This communication describes the purification and characterization of argininosuccinate synthetase from human liver. By numerous criteria including electrophoresis in sodium dodecyl sulfate containing gels, electrophoresis in nondissociating gels, and analytical ultracentrifugation, the protein is homogeneous at a specific activity of 4.2 mumol/(min mg) assayed at 37 degrees C in the direction of argininosuccinate synthesis. The enzyme has a molecular weight of 183,000, as determined by gel filtration. Electrophoresis in the presence of sodium dodecyl sulfate yielded a single band migrating with an Rf corresponding to 43,000 daltons. Thus, the enzyme is considered to contain four subunits of identical molecular weight. The s20,w of the enzyme is 8.2 S. Antibodies were prepared in rabbits directed against the purified protein. These antibodies react specifically with argininosuccinate synthetase, as determined by electrophoretic analysis of the immunoadsorbed product from crude extracts of human liver. The human enzyme has very similar properties to those published for the beef and rat liver enzymes.     

Acetyl-coenzyme-A carboxylase of Candida Lipolytica. 1. Purification and properties of the enzyme.

Acetyl-coenzyme-A carboxylase has been isolated in homogeneous form from Candida lipolytica. The homogeneity of the enzyme preparation is evidenced by analytical ultracentrifugation, dodecyl-sulfate-polyacrylamide gel electrophoresis and Ouchterlony double-diffusion analysis. The purified enzyme exhibits a specific activity of 8.0 U/mg protein at 25 degrees C and contains 1 mol biotin/263000 g protein. The sedimentation coefficient (S20,W) of the enzyme is 18 S. It has been shown by dodecyl-sulfate-polyacrylamide gel electrophoresis that the enzyme possesses only one kind of subunit with a molecular weight of 230000. This finding, together with the biotin content, indicates that the C. lipolytica enzyme has a highly integrated subunit structure. The C. lipolytica enzyme is very labile, but is stabilized by glycerol. The enzyme is markedly activated by poly(ethyleneglycol), the activation being due principally to a decrease in the Km values for substrates. Even in the presence of this activator, the Km value for acetyl-CoA of the C. lipolytica enzyme is much higher than that of the enzyme from Saccharomyces cerevisiae and animal tissues. The C. lipolytica enzyme, unlike the enzyme from animal tissues, is not activated by citrate.     

Purification and some molecular properties of protein methylase II from equine erythrocytes

Protein methylase II (S-adenosyl-L-methionine: protein carboxyl-O-methyltransferase; E.C. 2.1.1.24) has been purified 28 000 fold from equine erythrocytes. The purified enzyme is homogeneous on polyacrylamide gel electrophoresis performed either in presence or in absence of SDS, and on analytical ultracentrifugation. It appears constituted of a single polypeptidic chain of a molecular weight very close to 25 000 Daltons. Other enzymatic properties of the protein are quite similar to those previously reported for similar enzymes. The amino acid analysis of the enzyme is presented. The single cysteine residue, the enzyme contains, is essential for the enzymatic activity. Other amino acids apparentely involved in catalysis are tentatively identified.     

Beta-glucuronidase of rat preputial gland. Crystallization, properties, carbohydrate composition, and subunits.

Rat preputial gland beta-glucuronidase [ED 3.2.1.31] was purified by ammonium sulfate precipitation, ethanol fractionation, gel filtration on Sephadex G-200 and crystallization. The purified enzyme appeared homogeneous on electrophoresis in polyacrylamide gel, and on analytical ultracentrifugation and had a molecular weight of approximately 320,000, and a sedimentation coefficient of 12S. SDS polyacrylamide gel electrophoresis indicated that the enzyme consisted of subunits with molecular weight of 79,000, so the native enzyme appeared to be a tetramer. The Km with p-nitrophenyl beta-D-glucosiduronic acid as substrate was about 0.53 mM. The enzyme had a single pH optimum at 4.5. The enzyme had a very low content of sulphur-containing amino acid and contained 5.7 per cent carbohydrate, consisting of mannose, glucose, fucose, galactose, and glucosamine in a ratio of 44;9;6;2;41. Sialic acid was not detected in the crystallized enzyme.     

Purification and properties of a nonheme bromoperoxidase from Streptomyces aureofaciens

The first bacterial nonheme type bromoperoxidase has been purified to homogeneity from the chlorotetracycline-producing actinomycete Streptomyces aureofaciens Tü 24. Purification was accomplished by (NH4)2SO4 precipitation, DEAE-cellulose chromatography at different pH-values, and molecular sieve chromatography. The purified enzyme has a molecular mass of 90 to 95 kDa based on ultracentrifugation and gel filtration. The enzyme is composed of three subunits of identical molecular mass (m = 31 kDa). Bromoperoxidase catalyses the bromination of monochlorodimedone, but not its chlorination, and has no peroxidase or catalase activity. The optimum pH is 4.5. The enzyme does not exhibit an absorption peak in the Soret region of the optical spectrum. X-ray fluorescence spectroscopy revealed that the enzyme does not contain any metals in equimolar amounts. Bromoperoxidase is stable in a pH range from pH 4.0 to pH 10.0 at 4 degrees C for weeks and does not loose any activity when incubated at 80 degrees C for 2 h.     

Functional expression, purification, and characterization of the extra stable human placental alkaline phosphatase in the Pichia pastoris system

Human placental alkaline phosphatase was successfully cloned in the yeast system Pichia pastoris. The recombinant enzyme was over-expressed as a secreted protein in the cultured medium. The enzyme was extremely stable, which resulted in a total recovery of the enzyme activity after the purification process. The purified enzyme preparation was apparently homogeneous as examined by the polyacrylamide gel electrophoresis, analytical gel-permeation chromatography, and analytical ultracentrifugation. The final enzyme preparation showed a purification of 803-fold from the culture medium with a specific activity of 578 U/mg of protein. Fluorescence spectroscopic analyses showed multiple unfolding steps in the urea denaturation process of the homodimeric recombinant enzyme. Extensive conformational change of the enzyme in urea was detected by the analytical ultracentrifugation and the size-exclusive chromatography. The quaternary structure of the enzyme is quite stable. No indication of dissociation was observed after extensive tertiary structural changes.     

Purification and characterization of human plasma lecithin:cholesterol acyltransferase.

A highly purified (approximately 12 000-fold) homogeneous preparation of human plasma lecithin:cholesterol acyltransferase (LCAT) with 16% yield was obtained by a combination of density ultracentrifugation, high density lipoprotein affinity column chromatography, hydroxylapatite chromatography, and finally chromatography on anti-apolipoprotein D immunoglobulin-Sepharose columns to remove apolipoprotein D. This enzyme preparation was homogeneous by the following criteria: a single band by polyacrylamide gel electrophoresis in 8 M urea; a single band on sodium dodecyl sulfate gel electrophoresis with an apparent molecular weight of 68 000 +/- 1600; a single protein peak with a molecular weight of 70 000 on a calibrated Sephadex G-100 column. Its amino acid composition was different from human serum albumin and all other apoproteins isolated from lipoprotein fractions.     

Extracellular enzyme system utilized by the fungus Sporotrichum pulverulentum (Chrysosporium lignorum) for the breakdown of cellulose. 3. Purification and physico-chemical characterization of an exo-1,4-beta-glucanase.

An exo-1,4-beta-glucanase from culture solution of the rot fungus Sporotrichum pulverulentum (formerly called Chrysosporium lignorum) grown on powder cellulose as the sole carbon source has been extensively purified and characterized with respect to some physico-chemical properties. The purification has been carried out in a five-step procedure comprising chromatography on DEAE-Sephadex, gel filtration on polyacrylamide P-150, activation on a Dowex 2-X8 anion exchanger, chromatography on Concanavalin A-Sepharose and chromatography on SP-Sephadex. The purified enzyme was found to be pure and homogeneous by analytical polyacrylamide electrophoresis, by electrophoresis on dodecylsulphate gels and by analytical polyacrylamide electrophoresis, by electrophoresis on dodecylsulphate gels and by analytical isoelectric focusing. A single symmetrical peak was obtained with the free zone electrophoresis method. The purification factor is about 15 and the yield of exo-1,4-beta-glucanase activity 7%. After purification, the enzyme showed no viscosity-decreasing activity towards carboxymethyl-cellulose solutions. The exo-1,4-beta-glucanase was isoelectric at pH 4.3 (4 degrees C). A molecular weight of 48600 was calculated on the basis of a knowledge of the partial specific volume, ultracentrifugation data and the amino acid composition. The enzyme contained no carbohydrate.