Isolation and characterization of a xylose-glucose isomerase from a new strain Streptomyces thermovulgaris 127, var. 7-86

2001 ◽  
Vol 79 (2) ◽  
pp. 195-205 ◽  
Author(s):  
Vessela Raykovska ◽  
Pavlina Dolashka-Angelova ◽  
Donka Paskaleva ◽  
Stanka Stoeva ◽  
Juri Abashev ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Analysis ◽  
Protein Denaturation ◽  
Specific Activity ◽  
Guanidine Hydrochloride ◽  
Glucose Isomerase ◽  
Protein Sequencing ◽  
Γ Irradiation ◽  
Ph Optimum ◽  
Isolation And Characterization

A thermostable D-xylose–glucose isomerase was isolated from the thermophilic strain Streptomyces thermovulgaris 127, var. 7-86, as a result of mutagenic treatment by γ-irradiation of the parent strain, by precipitation and sequential chromatographies on DEAE–Sephadex A50, TSK-gel, FPLC-Mono Q/HR, and Superose 12™ columns. The N-terminal amino acid sequence and amino acid analysis shows 73–92% homology with xylose–glucose isomerases from other sources. The native molecular mass, determined by gel filtration on a Superose 12™ column, is 180 kDa, and 44.6 and 45 kDa were calculated, based on amino acid analysis and 10% SDS-PAGE, respectively. Both, the activity and stability of the enzyme were investigated toward pH, temperature, and denaturation with guanidine hydrochloride. The enzyme activity showed a clear pH optimum between pH 7.2 and 9.0 with D-glucose and 7.4 and 8.3 with D-xylose as substrates, respectively. The enzyme is active up to 60–85°C at pH 7.0, using D-glucose, and up to 50–60°C at pH 7.6, using D-xylose as substrates. The activation energy (Ea = 46 kJ·mol–1) and the critical temperature (Tc = 60°C) were determined by fluorescence spectroscopy. Tc is in close coincidence with the melting temperature of denaturation (Tm = 59°C), determined by circular dichroism (CD) spectroscopy. The free energy of stabilization in water after denaturation with Gdn.HCl was calculated to be 12 kJ·mol–1. The specific activity (km values) for D-xylose-glucose isomerase at 70°C toward different substrates, D-xylose, D-glucose, and D-ribose, were determined to be 4.4, 55.5, and 13.3 mM, recpectively.Key words: D-xylose-glucose isomerase, protein sequencing, protein stability, protein denaturation.

scholarly journals Thermostable glucose isomerase from psychrotolerant Streptomyces species

1970 ◽  
Vol 1 ◽  
pp. 6-10 ◽  
Author(s):  
Bidur Dhungel ◽  
Manoj Subedi ◽  
Kiran Babu Tiwari ◽  
Upendra Thapa Shrestha ◽  
Subarna Pokhrel ◽  
...  
Keyword(s):  
Specific Activity ◽  
Fold Increase ◽  
Glucose Isomerase ◽  
Enzyme Concentration ◽  
Maximal Velocity ◽  
Ph Optimum ◽  
Streptomyces Spp ◽  
Optimum Ph ◽  
Optimum Reaction ◽  
Sepharose 4B

Glucose isomerase (EC 5.3.1.5) was extracted from Streptomyces spp., isolated from Mt. Everest soil sample, and purified by ammonium sulfate fractionation and Sepharose-4B chromatography. A 7.1 fold increase in specific activity of the purified enzyme over crude was observed. Using glucose as substrate, the Michaelis constant (KM<) and maximal velocity (Vmax) were found to be 0.45M and 0.18U/mg. respectively. The optimum substrate (glucose) concentration, optimum enzyme concentration, optimum pH, optimum temperature, and optimum reaction time were 0.6M, 62.14μg/100μl, 6.9, 70ºC, and 30 minutes, respectively. Optimum concentrations of Mg2+ and Co2+ were 5mM and 0.5mM, respectively. The enzyme was thermostable with half-life 30 minutes at 100ºC.DOI: 10.3126/ijls.v1i0.2300 Int J Life Sci 1 : 6-10

Phosphoglycollate Phosphatase in Marine Algae: Isolation and Characterization from Halimeda cylindracea

1976 ◽  
Vol 3 (1) ◽  
pp. 105 ◽  
Author(s):  
DD Randall
Keyword(s):  
Divalent Cation ◽  
Marine Algae ◽  
Marine Alga ◽  
Specific Activity ◽  
Ph Optimum ◽  
Isolation And Characterization

Phosphoglycollate phosphatase was partially purified (107-fold) from the marine alga H. cylindracea. The partially purified enzyme was almost completely specific for phosphoglycollate as substrate, and had the following properties: a specific activity of 2.73, an apparent Km for phosphoglycollate of 8 x 10-4M, a pH optimum of 7.5 - 8 and a divalent cation requirement. The presence of the enzyme is discussed in relation to photorespiration in this alga.

scholarly journals Isolation and Characterization of a Soluble NADPH-Dependent Fe(III) Reductase from Geobacter sulfurreducens

2001 ◽  
Vol 183 (15) ◽  
pp. 4468-4476 ◽  
Author(s):  
Franz Kaufmann ◽  
Derek R. Lovley
Keyword(s):  
Amino Acid ◽  
Electron Donor ◽  
Formate Dehydrogenase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Amino Acid Sequences ◽  
Geobacter Sulfurreducens ◽  
Oxidoreductase Activity ◽  
Isolation And Characterization ◽  
Apparent Homogeneity

ABSTRACT NADPH is an intermediate in the oxidation of organic compounds coupled to Fe(III) reduction in Geobacter species, but Fe(III) reduction with NADPH as the electron donor has not been studied in these organisms. Crude extracts of Geobacter sulfurreducens catalyzed the NADPH-dependent reduction of Fe(III)-nitrilotriacetic acid (NTA). The responsible enzyme, which was recovered in the soluble protein fraction, was purified to apparent homogeneity in a four-step procedure. Its specific activity for Fe(III) reduction was 65 μmol · min−1 · mg−1. The soluble Fe(III) reductase was specific for NADPH and did not utilize NADH as an electron donor. Although the enzyme reduced several forms of Fe(III), Fe(III)-NTA was the preferred electron acceptor. The protein possessed methyl viologen:NADP+ oxidoreductase activity and catalyzed the reduction of NADP+ with reduced methyl viologen as electron donor at a rate of 385 U/mg. The enzyme consisted of two subunits with molecular masses of 87 and 78 kDa and had a native molecular mass of 320 kDa, as determined by gel filtration. The purified enzyme contained 28.9 mol of Fe, 17.4 mol of acid-labile sulfur, and 0.7 mol of flavin adenine dinucleotide per mol of protein. The genes encoding the two subunits were identified in the complete sequence of the G. sulfurreducens genome from the N-terminal amino acid sequences derived from the subunits of the purified protein. The sequences of the two subunits had about 30% amino acid identity to the respective subunits of the formate dehydrogenase from Moorella thermoacetica, but the soluble Fe(III) reductase did not possess formate dehydrogenase activity. This soluble Fe(III) reductase differs significantly from previously characterized dissimilatory and assimilatory Fe(III) reductases in its molecular composition and cofactor content.

The purification and characterization of a phospholipase A2 activity from the 106 000 × g pellet (microsomal fraction) of bovine brain acting on phosphatidylinositol

1982 ◽  
Vol 60 (2) ◽  
pp. 108-117 ◽  
Author(s):  
N. C. C. Gray ◽  
K. P. Strickland
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Microsomal Fraction ◽  
Sodium Dodecyl ◽  
Fatty Acid Distribution ◽  
Phospholipase A ◽  
Strong Inhibition ◽  
Ph Optimum ◽  
Triton X

A phospholipase A2 acting on phosphatidylinositol (PI) has been purified from the 106 000 × g pellet (microsomal fraction) of bovine grey matter. The purification steps included extraction with Triton X-100 (0.05%), ammonium sulfate fractionation (20–50% fraction), consecutive column chromatographic runs on Sephadex G-200 and DEAE-Sephacel, and preparative gel electrophoresis (on 10.5% polyacrylamide gel). These steps achieved a purification of 1614 times. The purified enzyme ran as a single band on sodium dodecyl sulfate (SDS) gel electrophoresis. Molecular weight estimations gave values of 18 300 by SDS gel electrophoresis and 18 521 based on amino acid analysis. Amino acid analysis showed the presence of 173 residues with aspartic acid (46), glutamic acid (26) and glycine (21) being the most abundant. Single residues of cysteine, tyrosine, and arginine were measured. The remaining 11 amino acids were present in amounts ranging from 3 to 11 residues.The purified enzyme had a pH optimum of 7.4, was heat stable (to 70 °C), and was activated by Ca2+ (5 mM). Other divalent cations were either slightly inhibitory (Mg2+ and Mn2+) or strongly inhibitory (Zn2+). The nonionic detergents, Triton X-100 (0.02 to 0.03%) and octyl glucoside (30 mM) showed 70 and 25% stimulations, respectively. Other detergents showed no effect (Cutscum), slight inhibition (G3634A), or strong inhibition (cetyltrimethylammonium bromide). Determination of the apparent Km and Vmax by an Eisthenal–Cornish-Bowden plot gave values of 0.52 mM and 1440 nmol [1-14C]oleic acid min −1∙mg protein −1, respectively, for 1-acyl-2-[1-,14C]oleoyl-sn glycerol-3-phosphoinositol as substrate. The above plot confirmed the presence of a strong inhibition by substrate (i.e., PI) beyond 0.4 mM. The properties of this enzyme and its location (microsomal) make it uniquely different from other phospholipase A2 activities reported for brain. The microsomal location and preference for PI shown by this enzyme lend support to the view that it may function to form lyso-PI in a deacylation–reacylation cycle for altering the fatty acid distribution in PI.

scholarly journals Purification, characterization, DNA sequence and cloning of a pimeloyl-CoA synthetase from Pseudomonas mendocina 35

1999 ◽  
Vol 340 (3) ◽  
pp. 793-801 ◽  
Author(s):  
Andrew BINIEDA ◽  
Martin FUHRMANN ◽  
Bruno LEHNER ◽  
Claudine REY-BERTHOD ◽  
Séverine FRUTIGER-HUGHES ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Sequence ◽  
Specific Activity ◽  
Amino Acid Sequences ◽  
Active Enzyme ◽  
Gene Probe ◽  
Pimelic Acid ◽  
Pseudomonas Mendocina ◽  
Ph Optimum ◽  
E Coli

A pimeloyl-CoA synthetase from Pseudomonas mendocina 35 was purified and characterized, the DNA sequence determined, and the gene cloned into Escherichia coli to yield an active enzyme. The purified enzyme had a pH optimum of ≈ 8.0, Km values of 0.49 mM for pimelic acid, 0.18 mM for CoA and 0.72 mM for ATP, a subunit Mr of ≈ 80000 as determined by SDS/PAGE, and was found to be a tetramer by gel-filtration chromatography. The specific activity of the purified enzyme was 77.3 units/mg of protein. The enzyme was not absolutely specific for pimelic acid. The relative activity for adipic acid (C6) was 72% and for azaleic acid (C9) was 18% of that for pimelic acid (C7). The N-terminal amino acid was blocked to amino acid sequencing, but controlled proteolysis resulted in three peptide fragments for which amino acid sequences were obtained. An oligonucleotide gene probe corresponding to one of the amino acid sequences was synthesized and used to isolate the gene (pauA, imelic cid-tilizing ) coding for pimeloyl-CoA synthetase. The pauA gene, which codes for a protein with a theoretical Mr of 74643, was then sequenced. The deduced amino acid sequence of the enzyme showed similarity to hypothetical proteins from Archaeoglobus fulgidus, Methanococcus jannaschii, Pyrococcus horikoshii, E. coli and Streptomyces coelicolor, and some limited similarity to microbial succinyl-CoA synthetases. The similarity with the protein from A. fulgidus was especially strong, thus indicating a function for this unidentified protein. The pauA gene was cloned into E. coli, where it was expressed and resulted in an active enzyme.

scholarly journals Isolation, amino acid analyses and refolding of subunits of pig heart succinyl-CoA synthetase

1988 ◽  
Vol 250 (2) ◽  
pp. 429-434 ◽  
Author(s):  
J S Nishimura ◽  
J Ybarra ◽  
T Mitchell ◽  
P M Horowitz
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
Guanidine Hydrochloride ◽  
Beta Subunit ◽  
Native Enzyme ◽  
Tryptophan Residue ◽  
Beta Subunits ◽  
Post Translational Modification ◽  
E Coli ◽  
Succinyl Coa Synthetase

For the first time, pig heart succinyl-CoA synthetase has been refolded from its isolated subunits after denaturation. Amino acid analyses of pig heart succinyl-CoA synthetase and its subunits were performed. Subunits were isolated by gel filtration in neutral 6 M-urea. The amino acid composition of the native enzyme bears a strong resemblance to that of the Escherichia coli enzyme. Application of the various methods for comparing amino acid compositions [Cornish-Bowden (1983) Methods Enzymol. 91, 60-75] shows that the degree of relatedness between the alpha-subunits of the pig heart and E. coli enzymes and between the beta-subunits of the two synthetases is intermediate between ‘strong’ and ‘weak’. As for the E. coli synthetase, it is unlikely that the alpha-subunit arises from the larger beta-subunit by post-translational modification. The pig heart enzyme contains a single tryptophan residue, which is located in the beta-subunit. Excitation of the enzyme at 295 nm resulted in a typical tryptophan emission spectrum. Refolding of enzyme denatured in 6 M-guanidine hydrochloride or of alpha- and beta-subunits isolated in this solvent required the presence of either ethylene glycol or glycerol, optimally at 20-25% (v/v). GTP-Mg2+ did not stimulate reactivation of the enzyme, in contrast with the result obtained with ATP-Mg2+ in the reconstitution of the enzyme from E. coli. Yields of 60% and 40% were obtained in the refolding of denatured enzyme and isolated subunits respectively. The fluorescence spectrum of the refolded protein was essentially the same as that of native enzyme. Unrecovered activity could not be accounted for in the form of protein aggregates. The specific activity of refolded enzyme that had been separated from inactive protein on a Bio-Sil TSK 250 column was the same as that of native enzyme. Km values for GTP of 27 microM and 14 microM were determined for native and refolded enzyme respectively.

Isolation and characterization of human LH isoforms

1993 ◽  
Vol 138 (3) ◽  
pp. 529-543 ◽  
Author(s):  
P. G. Stanton ◽  
G. Pozvek ◽  
P. G. Burgon ◽  
D. M. Robertson ◽  
M. T. W. Hearn
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Amino Acid Analysis ◽  
High Performance ◽  
Purification Procedure ◽  
Human Pituitary ◽  
Isolation And Characterization ◽  
Reducing Conditions ◽  
Sds Page ◽  
Α And Β Subunits

ABSTRACT Thirty-nine human LH (hLH) isoforms were chromatographically separated from human pituitary extracts using a mild purification procedure which consisted of preparative isoelectric focusing, high-performance ion-exchange chromatography and immobilized metal-affinity chromatography. Twenty of these hLH isoforms were characterized by LH radioreceptor assay, SDS-PAGE and amino acid analysis, and 17 were shown to be highly purified (>90% pure). The specific activities of these hLH isoforms ranged from 1980 to 38 650 IU/mg protein in terms of the 2nd IS for human pituitary LH, based on protein content as determined by amino acid analysis. hFSH and hTSH content were <0·5% and <7·8% respectively. The purity was assessed by silver staining on SDS-PAGE. Under non-reducing conditions, a single band of apparent molecular mass 23·5–24·5 kDa was observed, whereas under reducing conditions the isoforms migrated as two distinct bands, 21·1–22·4 kDa and 18·0–20·5 kDa, probably corresponding to the α and β subunits of hLH respectively. The remaining three less pure isoform preparations (70–90% pure) contained additional bands of 16 kDa and 26·3 kDa under non-reducing conditions. All isoforms showed a low molecular mass band(s) of 11–14 kDa which was <7% of stained material as assessed by densitometry. Amino acid composition of the 17 hLH isoforms was similar to the published cDNA composition of hLH. Further fractionation of one hLH isoform (hLH IIc) on reversed-phase high-performance liquid chromatography yielded four peaks identified by N-terminal sequencing as two α and two β hLH subunits identical to their cDNA-derived N-terminal sequences. No additional sequences indicative of internal clipping of hLH were observed. The two pairs of α and β subunits probably represent two separate hLH isoforms in this preparation. It was concluded that a mild purification procedure with high recoveries for the isolation of intact hLH isoforms has been developed, and 17 isoforms of high purity suitable for further biological and physicochemical characterization have been isolated. These isoform preparations are free of other contaminating proteins, but may still contain multiple hLH-related species. Journal of Endocrinology (1993) 138, 529–543

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