scholarly journals Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway

2005 ◽  
Vol 387 (1) ◽  
pp. 271-280 ◽  
Author(s):  
Seonghun KIM ◽  
Sun Bok LEE
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Genome Database ◽  
Sds Page ◽  
Key Enzyme ◽  
Bivalent Metal Ions ◽  
Ammonium Sulphate Fractionation ◽  
Identification And Characterization

The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner–Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 °C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 °C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with Km=0.45 mM and Vmax=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation–dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner–Doudoroff pathway.

scholarly journals Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

1999 ◽  
Vol 181 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Hisayo Ono ◽  
Kazuhisa Sawada ◽  
Nonpanga Khunajakr ◽  
Tao Tao ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Mass ◽  
Optimum Temperature ◽  
Sds Page ◽  
Halomonas Elongata ◽  
Optimum Ph

ABSTRACT 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had Km s of 9.1 mM forl-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.

scholarly journals Purification and characterization of a thermostable ADP-glucose pyrophosphorylase from Thermus caldophilus GK-24

1996 ◽  
Vol 319 (3) ◽  
pp. 977-983 ◽  
Author(s):  
Jeong Heon KO ◽  
Cheorl Ho KIM ◽  
Dae-Sil LEE ◽  
Yu Sam KIM
Keyword(s):  
Molecular Mass ◽  
Specific Activity ◽  
Gel Filtration ◽  
Higher Plant ◽  
Sds Page ◽  
Thermus Caldophilus ◽  
Internal Peptide ◽  
Adp Glucose Pyrophosphorylase ◽  
Fructose 1,6 Bisphosphate

An extremely thermostable ADP-glucose pyrophosphorylase (AGPase) has been purified from Thermus caldophilus GK-24 to homogeneity by chromatographic methods, including gel filtration and ion-exchange and affinity chromatography. The specific activity of the enzyme was enriched 134.8-fold with a recovery of 10.5%. The purified enzyme was a single band by SDS/PAGE with a molecular mass of 52 kDa. The homotetrameric structure of the native enzyme was determined by gel filtration analysis, which showed a molecular mass of 230 kDa on a Superose-12 column, indicating that the structure of the enzyme is different from the heterotetrameric structures of higher-plant AGPases. The enzyme was most active at pH 6.0. The activity was maximal at 73–78 °C and its half-life was 30 min at 95 °C. Kinetic and regulatory properties were characterized. It was found that AGPase activity could be stimulated by a number of glycolytic intermediates. Fructose 6-phosphate, fructose 1,6-bisphosphate, phenylglyoxal and glucose 6-phosphate were effective activators, of which fructose 1,6-bisphosphate was the most effective. The enzyme was inhibited by phosphate, AMP or ADP. ATP and glucose 1-phosphate gave hyperbolic-shaped rate-concentration curves in the presence or absence of activator. A remarkable aspect of the amino acid composition was the existence of the hydrophobic and Ala+Gly residues. The N-terminal and internal peptide sequences were determined and compared with known sequences of various sources. It was apparently similar to those of AGPases from other bacterial and plant sources, suggesting that the enzymes are structurally related.

Production, purification, and characterization of an extracellular endo-β-1, 3-glucanase from a monokaryon of Schizophyllum commune ATCC 38548 defective in exo-β-1, 3-glucanase formation

1994 ◽  
Vol 40 (1) ◽  
pp. 18-23 ◽  
Author(s):  
Andreas Prokop ◽  
Peter Rapp ◽  
Fritz Wagner
Keyword(s):  
Molecular Mass ◽  
Schizophyllum Commune ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Glucanase Activity ◽  
Sds Page ◽  
S 100 ◽  
Reduced Activity

Production of extracellular β-1, 3-glucanase activity by a monokaryotic Schizophyllum commune strain was monitored and results indicated that the β-glucanase activity consisted of an endo- β-1, 3-glucanase activity, besides a negligible amount of β-1, 6-glucanase and β-glucosidase activity. Unlike the β-1, 3-glucanase production of the dikaryotic parent strain S. commune ATCC 38548, the β-1, 3-glucanase formation of the monokaryon was not regulated by catabolite repression. The endo- β-1, 3-glucanase of the monokaryon was purified from the culture filtrate by lyophilization, anion exchange chromatography on Mono Q, and gel filtration on Sephacryl S-100. It appeared homogeneous on SDS-PAGE with a molecular mass of 35.5 kDa and the isoelectric point was 3.95. The enzyme was only active toward glucans containing β-1, 3-linkages, including lichenan, a β-1, 3-1, 4-D-glucan. It attacked laminarin in an endo-like fashion to form laminaribiose, laminaritriose, and high oligosaccharides. While the extracellular β-glucanases from the dikaryotic S. commune ATCC 38548 degraded significant amounts of schizophyllan, the endo- β-1, 3-glucanase from the monokaryon showed greatly reduced activity toward this high molecular mass β-1, 3-/β-1, 6-glucan. The Km of the endoglucanase, using laminarin as substrate, was 0.28 mg/mL. Optimal pH and temperature were 5.5 and 50 °C, respectively. The enzyme was stable between pH 5.5 and 7.0 and at temperatures below 50 °C. The enzyme was completely inhibited by 1 mM Hg2+. Growth of the monokaryotic S. commune strain was not affected by its constitutive endo- β-1, 3-glucanase formation.Key words: endo- β-1, 3-glucanase, Schizophyllum commune, monokaryon, constitutive endo- β-1, 3-glucanase formation.

scholarly journals Purification and characterization of an extracellular (1 → 6)-β-glucanase from the filamentous fungus Acremonium persicinum

1996 ◽  
Vol 316 (3) ◽  
pp. 841-846 ◽  
Author(s):  
Stuart M. PITSON ◽  
Robert J. SEVIOUR ◽  
Barbara M. McDOUGALL ◽  
Bruce A. STONE ◽  
Maruse SADEK
Keyword(s):  
Molecular Mass ◽  
Filamentous Fungus ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Eisenia Bicyclis ◽  
Ph Optimum ◽  
Hydrolysis Products ◽  
Sds Page ◽  
Hydrolysis Of

An endo-(1 → 6)-β-glucanase has been isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. SDS/PAGE of the purified enzyme gave a single band with an apparent molecular mass of 42.7 kDa. The enzyme is a non-glycosylated, monomeric protein with a pI of 4.9 and pH optimum of 5.0. It hydrolysed (1 → 6)-β-glucans (pustulan and lutean), initially yielding a series of (1 → 6)-β-linked oligoglucosides, consistent with endo-hydrolytic action. Final hydrolysis products from these substrates were gentiobiose and gentiotriose, with all products released as β-anomers, indicating that the enzyme acts with retention of configuration. The purified enzyme also hydrolysed Eisenia bicyclis laminarin, liberating glucose, gentiobiose, and a range of larger oligoglucosides, through the apparent hydrolysis of (1 → 6)-β- and some (1 → 3)-β-linkages in this substrate. Km values for pustulan, lutean and laminarin were 1.28, 1.38, and 1.67 mg/ml respectively. The enzyme was inhibited by N-acetylimidazole, N-bromosuccinimide, dicyclohexylcarbodi-imide, Woodward's Regent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4 and some metal ions, whereas D-glucono-1,5-lactone and EDTA had no effect.

scholarly journals Purification and characterization of casein kinase I from broccoli

1993 ◽  
Vol 293 (1) ◽  
pp. 283-288 ◽  
Author(s):  
L J Klimczak ◽  
A R Cashmore
Keyword(s):  
Molecular Mass ◽  
Specific Activity ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Casein Kinase ◽  
Casein Kinase I ◽  
Native Molecular Mass ◽  
Specificity And Sensitivity ◽  
Sds Page

Casein kinase I from broccoli was purified approximately 65,000-fold by chromatography on phosphocellulose, phenyl-Sepharose, CM-Sephacel, and affinity chromatography on N-(2-aminoethyl)-5-chloroisoquinolone-8-sulphonamide (CKI-7)-Sepharose. The catalytic subunit of casein kinase I was identified as a 36-38 kDa polypeptide doublet by using the technique of activity gel assay after SDS/PAGE with casein as a gel-incorporated substrate. A silver-stained polypeptide doublet of the same molecular mass constituted at least 95% of the protein in the final preparation, corresponding to a specific activity of approximately 1800 nmol/min per mg of protein. The enzyme was found to be a monomer by gel filtration and glycerol gradient sedimentation; the native molecular mass was calculated to be 34.2 kDa. These characteristics, as well as other essential features of plant casein kinase I activity, such as substrate specificity and sensitivity to inhibitors, were found to be similar to those established for animal casein kinase I. Broccoli casein kinase I showed weak immunological cross-reactivity with antibodies raised against bovine casein kinase I.

scholarly journals Characterization of the hydroxymethylglutaryl-CoA lyase precursor, a protein targeted to peroxisomes and mitochondria

1996 ◽  
Vol 315 (1) ◽  
pp. 71-75 ◽  
Author(s):  
Lyudmila I. ASHMARINA ◽  
Marie-France ROBERT ◽  
Marc-André ELSLIGER ◽  
Grant A. MITCHELL
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Leader Peptide ◽  
Mitochondrial Targeting ◽  
Ph Optimum ◽  
Sds Page ◽  
Peroxisomal Targeting ◽  
Quaternary Structures ◽  
Hydroxymethylglutaryl Coa Lyase

We previously showed that human liver hydroxymethylglutaryl-CoA (HMG-CoA) lyase (HL; EC 4.1.3.4) is found in both mitochondria and peroxisomes. HL contains a 27-residue N-terminal mitochondrial targeting sequence which is cleaved on mitochondrial entry, as well as a C-terminal Cys-Lys-Leu peroxisomal targeting motif. Because peroxisomal HL has a greater molecular mass and more basic pI value than mitochondrial HL, we predicted that peroxisomal HL retains the mitochondrial leader. To test this hypothesis, we expressed both the precursor (pHL) and mature (mHL) peptides in Escherichia coli and studied their properties. pHL purified by ion-exchange and hydrophobic chromatography had a pI of 7.6 on FPLC chromatofocusing and a molecular mass of 34.5 kDa on SDS/PAGE, similar to our findings for peroxisomal HL. For purified mHL, pI (6.2) and molecular mass (32 kDa) values resemble those of mitochondrial HL. Purified pHL is similar to mHL in Km for HMG-CoA (44.8 μM), kcat (6.3 min-1) and pH optimum (9.0–9.5). However, the quaternary structures of pHL and mHL differ. On Superose 12 FPLC gel filtration and also on ultrafiltration, both in the presence and in the absence of HMG-CoA, pHL behaves as a monomer whereas mHL migrates as a dimer. We conclude that the HL precursor is probably identical to peroxisomal HL, that its catalytic properties resemble those of mature mitochondrial HL, and that the mitochondrial leader peptide prevents dimerization of pHL.

Purification and Characterization of Alkaline Polygalacturonases Produced by Paenibacillus polymyxa 20185 in Submerged Cultures

2012 ◽  
Vol 550-553 ◽  
pp. 1496-1502
Author(s):  
Hong Ju Jiang ◽  
Wei Zhen Xu ◽  
Jia Mei Song ◽  
Dong Ping Sun
Keyword(s):  
Gel Filtration ◽  
Paenibacillus Polymyxa ◽  
Maximal Activity ◽  
Purification And Characterization ◽  
Submerged Cultures ◽  
Liquid Cultures ◽  
Similar Molecular Weight ◽  
Sds Page ◽  
Alkaline Conditions

Two extracellular alkaline polygalacturonases from extracts of liquid cultures of Paenibacillus polymyxa 20185 were purified by gel filtration chromatography to homogeneity as judged by SDS-PAGE. The purified alkaline polygalacturonases (PG1 and PG2) had a similar molecular weight of 65 kDa, exhibited maximal activity at 50°C with pH 10.0, and were stable in alkaline conditions. The purified alkaline polygalacturonases activities were enhanced in the presence of Mg2+, and were resistant to inhibition by Mn2+, Zn2+and Cu2+. Michaelis-menten constants of PG1 and PG2 were found as 3.6mg/mL and 3.5mg/mL, respectively.

scholarly journals Production, purification and characterization of an exo-polygalacturonase from Penicillium janthinellum sw09

2016 ◽  
Vol 88 (suppl 1) ◽  
pp. 479-487 ◽  
Author(s):  
YUPING MA ◽  
SIWEN SUN ◽  
HUI HAO ◽  
CHUNPING XU
Keyword(s):  
Gel Filtration ◽  
Maximal Activity ◽  
Growth Conditions ◽  
Purification And Characterization ◽  
Penicillium Janthinellum ◽  
Soil Isolate ◽  
Sds Page ◽  
The Stability ◽  
Ph Range

ABSTRACT A soil isolate, Penicillium janthinellum sw09 has been found to produce significant amounts of an extracellular pectinase subsequently characterized as exo-polygalacturonase (exo-PG). By optimizing growth conditions, P. janthinellum sw09 produced high amount of exo-PG (16.54 units/mL). The crude enzyme was purified by gel filtration chromatography and two exo-PG activity peaks (designated as PGI and PGII) were revealed. On SDS-PAGE analysis, purified PGII using DEAE-Sepharose FF column, was found to be a single band with a molecular mass of 66.2 kDa. The purified PGII exhibited maximal activity at the temperature of 45 oC and pH 5.0. The stability profiles show that PGII is more stable in the pH range of 4.0-8.0 and below 60 oC. The Km and Vmax for the enzyme was 1.74 mg/mL and 18.08 μmol/ (mL•min), respectively. Due to this enzymatic characterization, this pectinase is an attractive candidate for applications in degradation of pectin.

Purification and partial biochemical–genetic characterization of trehalose 6-phosphate synthase from muscles of adult femaleAscaris suum

2012 ◽  
Vol 87 (2) ◽  
pp. 212-221 ◽  
Author(s):  
M. Dmitryjuk ◽  
M. Dopieralska ◽  
E. Łopieńska-Biernat ◽  
R.J. Frączek
Keyword(s):  
Genetic Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sds Page ◽  
Optimum Ph ◽  
Genes Encoding ◽  
Ammonium Sulphate Fractionation ◽  
Biochemical Genetic

AbstractTrehalose 6-phosphate (T6P) synthase (TPS;EC2.4.1.15) was isolated from muscles ofAscaris suumby ammonium sulphate fractionation, ion-exchange DEAE SEPHACELTManion exchanger column chromatography and Sepharose 6B gel filtration. On sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), 265-fold purified TPS exhibited a molecular weight of 66 kDa. The optimum pH and temperature of the purified enzyme were 3.8–4.2 and 35°C, respectively. The isoelectric point (pI) of TPS was pH 5.4. The studied TPS was not absolutely substrate specific. Besides glucose 6-phosphate, the enzyme was able to use fructose 6-phosphate as an acceptor of glucose. TPS was activated by 10 mMMgCl2, 10 mMCaCl2and 10 mMNaCl. In addition, it was inhibited by ethylenediaminetetra-acetic acid (EDTA), KCl, FeCl3and ZnCl2. Two genes encoding TPS were isolated and sequenced from muscles of the parasite. Complete coding sequences fortps1(JF412033.2) andtps2(JF412034.2) were 3917 bp and 3976 bp, respectively. Translation products (AEX60788.1 and AEX60787.1) showed expression to the glucosyltransferase-GTB-type superfamily.

scholarly journals Purification and Characterization of Sepiapterin Deaminase from the Silkworm, Bombyx mori

Pteridines ◽  
1998 ◽  
Vol 9 (1) ◽  
pp. 18-21 ◽  
Author(s):  
Hiroshi Sawada ◽  
Motoki Kanekatsu ◽  
Motoko Nakagoshi ◽  
Kenjiro Dohke ◽  
Teruhiko Iino ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Bombyx Mori ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Native Form ◽  
Sds Page ◽  
Column Chromatographic ◽  
Silkworm Bombyx Mori

Summary Sepiapterin deaminase has been purified approximately 6,000-told from the larval integument of the lemon mutant of the silkworm by several column chromatographic procedures. Sepiapterin and isosepiapterin were active substrates among various pteridines tested. The molecular mass of this enzyme was estimated to be 74 kDa by SDS-PAGE and 70 kDa by gel filtration, suggesting that the native form of the enzyme is monomeric protein . All silkworm strains, to the best of our knowledge, had an activity of the enzyme and the enzyme was widely distributed in the larval tissues. Sepiapterin deaminase may have an important function on the silkworm.

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