Purification and physical properties of glucokinase from Thiobacillus versutus (A2)

1986 ◽  
Vol 32 (12) ◽  
pp. 937-941 ◽  
Author(s):  
David P. Klein ◽  
A. Michael Charles
Keyword(s):  
Physical Properties ◽  
Divalent Cation ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Optimum Temperature ◽  
Original Activity ◽  
Absolute Requirement ◽  
Thiobacillus Versutus ◽  
Optimum Ph ◽  
Temperature Ranges

Glucokinase was purified 749-fold from Thiobacillus versutus. Polyacrylamide gel electrophoresis revealed the presence of five protein bands in the purified preparation. The purified enzyme retained its original activity after 4 weeks of storage at −20 °C, but not at higher temperatures. Glucose provided some protection at 25 °C. The optimum temperature for activity was between 20 and 25 °C, and an energy of activation (Ea) of 1.305 kcal/mol was calculated. A Q10 value of about 1.080 was determined over two 10 °C temperature ranges between 5 and 20 °C. With both Hepes and Tricine buffers, the optimum pH was 7.8. The enzyme was specific for glucose, with a Km of 0.86 mM. The most efficient phosphoryl donor was ATP with a Km of 0.78 mM. About 30% of the activity observed with ATP was obtained with equimolar amounts of ITP, while TTP and UTP gave 7.0 and 12%, respectively. The enzyme displayed an absolute requirement for a divalent cation, with Mg2+ (Km, 0.27 mM) being the most effective.

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.

Extracellular endodextranase from the yeast Lipomyces starkeyi

1983 ◽  
Vol 29 (9) ◽  
pp. 1092-1095 ◽  
Author(s):  
E. Webb ◽  
I. Spencer-Martins
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Minimal Medium ◽  
Enzyme Preparation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Extracellular Fluid ◽  
Sole Source ◽  
Optimum Temperature ◽  
Lipomyces Starkeyi ◽  
High Concentrations

Strain IGC 4047 of the yeast Lipomyces starkeyi grew well with dextran as sole source of carbon and energy, and was able to hydrolyse blue dextran and Sephadex G-100. The enzyme was partially purified by fractionated isopropanol precipitation from the extracellular fluid of cultures grown in a minimal medium with dextran. The enzyme preparation showed only one band by polyacrylamide gel electrophoresis. The enzyme had the following properties: molecular weight, 23 000; optimum temperature and pH for activity, around 50 °C and pH 5.0, respectively; pH stability, pH 3.5–7.5; after 2 h at 50 °C and pH 5.0, 30% reduction in activity; isoelectric point, pI = 5.4; final products of dextran hydrolysis, isomaltooligosaccharides from glucose up to isomaltohexaose, with high concentrations of isomaltose and isomaltotriose. These results suggest that the enzyme is an endodextranase.

Purification and some properties of the extracellular α-amylase from Aspergillus awamori

1985 ◽  
Vol 31 (2) ◽  
pp. 149-153 ◽  
Author(s):  
Resham S. Bhella ◽  
Illimar Altosaar
Keyword(s):  
Enzyme Activity ◽  
Gel Electrophoresis ◽  
Culture Medium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Aspergillus Awamori ◽  
Original Activity

Alpha-amylase was purified from the extracellular culture medium of Aspergillus awamori by means of ethanol precipitation. Sephacryl-200 gel filtration and anion-exchange chromatography on Dowex (AG1-X4) resin. The enzyme preparation was found to be homogeneous by means of sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of 54 000 ± 2 500 and its isoelectric point was pH 4.2. The enzyme was found to be most active between pH 4.8 and 5.0 and was stable between pH 3.5 and 6.5. The optimal temperature for the enzyme activity was around 50 °C and the enzyme was stable for at least 1 h up to 45 °C retaining more than 80% of its original activity. The Km (37 °C, pH 5.3) for starch hydrolysis was 1.0 g∙L−1 and maltose inhibited the enzyme activity uncompetitively with a K1 value of 20.05 g∙L−1

β-D-Xylanases of Bacillus circulans WL-12

1982 ◽  
Vol 28 (7) ◽  
pp. 733-739 ◽  
Author(s):  
R. Esteban ◽  
J. R. Villanueva ◽  
T. G. Villa
Keyword(s):  
Gel Electrophoresis ◽  
Ph Value ◽  
Polyacrylamide Gel Electrophoresis ◽  
Negative Electrode ◽  
Polyacrylamide Gel ◽  
Bacillus Circulans ◽  
Michaelis Constant ◽  
Molecular Weights ◽  
Hydrolysis Products ◽  
Optimum Ph

Bacillus circulans WL-12 secretes two endo-β-D-xylanases (A and B, respectively) (EC 3.2.1.8.) and one β-D-xylosidase (EC 3.2.1.37) when cultured in liquid media with xylan as the sole carbon source. Xylanases A and B have been partially characterized with respect to their main physicochemical parameters and β-D-xylosidase to a lesser extent on account of its low stability. Both endo-β-D-xylanase A and β-D-xylosidase were adsorbed on DEAE-Biogel A, had similar molecular weights (approximately 85 000), and had optimum pH values of 5.5–7, but exhibited different isoelectric points (4.5 for β-D-xylanase A and 4.7 for β-D-xylosidase) and different mobilities in polyacrylamide gel electrophoresis. The apparent Michaelis constant for β-D-xylanase A was 8 mg∙mL−1 and the hydrolysis products produced were xylose, xylobiose, xylotriose, and xylotetraose.The second endo-β-D-xylanase (β-D-xylanase B) bound to CM-Biogel A and exhibited a molecular weight of approximately 15 000 and an optimum pH value in the range of 5.5–7. The isoelectric point was 9.1 and the apparent Michaelis constant was 4 mg∙mL−1. The hydrolysis products produced by this enzyme were xylobiose, xylotriose, and xylotetraose, but never xylose. In polyacrylamide gel electrophoresis at pH 8 the enzyme moved towards the negative electrode.

scholarly journals Characterization of a Thermostable d-Stereospecific Alanine Amidase from Brevibacillus borstelensis BCS-1

2003 ◽  
Vol 69 (2) ◽  
pp. 980-986 ◽  
Author(s):  
Dae Heoun Baek ◽  
Seok-Joon Kwon ◽  
Seung-Pyo Hong ◽  
Mi-Sun Kwak ◽  
Mi-Hwa Lee ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Optimum Temperature ◽  
Gene Encoding ◽  
Ph Range

ABSTRACT A gene encoding a new thermostable d-stereospecific alanine amidase from the thermophile Brevibacillus borstelensis BCS-1 was cloned and sequenced. The molecular mass of the purified enzyme was estimated to be 199 kDa after gel filtration chromatography and about 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme could be composed of a hexamer with identical subunits. The purified enzyme exhibited strong amidase activity towards d-amino acid-containing aromatic, aliphatic, and branched amino acid amides yet exhibited no enzyme activity towards l-amino acid amides, d-amino acid-containing peptides, and NH2-terminally protected amino acid amides. The optimum temperature and pH for the enzyme activity were 85°C and 9.0, respectively. The enzyme remained stable within a broad pH range from 7.0 to 10.0. The enzyme was inhibited by dithiothreitol, 2-mercaptoethanol, and EDTA yet was strongly activated by Co2+ and Mn2+. The k cat/Km for d-alaninamide was measured as 544.4 ± 5.5 mM−1 min−1 at 50°C with 1 mM Co2+.

scholarly journals Arginase from human full-term placenta

1976 ◽  
Vol 159 (3) ◽  
pp. 579-583 ◽  
Author(s):  
R Porta ◽  
C Esposito ◽  
A Martin ◽  
G D Pietra
Keyword(s):  
Gel Electrophoresis ◽  
Competitive Inhibition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Full Term ◽  
Term Placenta ◽  
A Value ◽  
Optimum Ph

Arginase was purified about 1800-fold from extracts of human full-term placenta; the enzyme appeared to be homogenous by disc electrophoresis and molecular-sieve chromatography. The mol. wt. determination by gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis yielded a value of 70000 for the most pure and the partially purified enzyme. The human placenta arginase is a metalloenzyme with an optimum pH of 9.1. The Km for L-arginine is 27 mM. L-Ornithine and L-lysine show competitive inhibition with Ki values of 6.3 and 14 mM respectively.

scholarly journals Purification and properties of α-D-mannosidase from the germinated seeds of Medicago sativa (alfalfa)

1980 ◽  
Vol 185 (2) ◽  
pp. 455-462 ◽  
Author(s):  
A Curdel ◽  
F Petek
Keyword(s):  
Medicago Sativa ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Purification Method ◽  
Purification And Properties ◽  
Optimum Ph ◽  
Citrate Phosphate

alpha-Mannosidase of Medicago sativa (alfalfa) was purified 1340-fold. The purification method included dialysis of the crude extract against a citrate/phosphate buffer, pH 3.9, (NH4)SO4 precipitation, hydroxyapatite chromatography, chromatography on Sephadex G-200 and finally a preparatory electrophoresis on polyacrylamide-gel gradient by Doly & Petek's [(1977) J. Chromatogr. 137. 69-81] method. Each step of purification was checked by polyacrylamide-gel disc electrophoresis. The purified enzyme showed a single band, corresponding to alpha-mannosidase activity. alpha-Mannosidase has a mol.wt. 230 000 as estimated by Hedrick & Smith's [(1968) Arch. Biochem. Biophys. 126, 155-164] method and also by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate by Weber & Osborn [(1969) J. Biol. Chem. 244, 4406-4412]. The enzyme comprises four subunits of different molecular weight. Optimum pH and Km values were determined with p-nitrophenyl alpha-D-mannoside as substrate. When incubated at a temperature between 20 and 62 degrees C before assay, alpha-mannosidase initially shows an increase in activity. alpha-Mannosidase is stable when the pH is about neutrality. It can be inactivated by several metal ions, including Zn2+. At a pH below 5 the enzyme undergoes irreversible inactivation. The presence of EDTA at acid pH considerably enhances the inactivation of the enzyme. This inactivation due to EDTA can be specifically reversed by incubation with Zn2+.

scholarly journals Purification and properties of Cellvibrio gilvus cellobiose phosphorylase

1983 ◽  
Vol 209 (3) ◽  
pp. 803-807 ◽  
Author(s):  
T Sasaki ◽  
T Tanaka ◽  
S Nakagawa ◽  
K Kainuma
Keyword(s):  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Competitive Inhibitor ◽  
Polyacrylamide Gel ◽  
Protamine Sulphate ◽  
Purification And Properties ◽  
Cellobiose Phosphorylase ◽  
Optimum Ph

The cellobiose phosphorylase (EC 2.4.1.20) of Cellvibrio gilvus, which is an endocellular enzyme, has been purified 196-fold with a recovery of 11% and a specific activity of 27.4 mumol of glucose 1-phosphate formed/min per mg of protein. The purification procedure includes fractionation with protamine sulphate, and hydroxyapatite and DEAE-Sephadex A-50 chromatography. The enzyme appears homogeneous on polyacrylamide-gel electrophoresis, and a molecular weight of 280 000 was determined by molecular-sieve chromatography. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed a single band and mol.wt. 72 000, indicating that cellobiose phosphorylase consists of four subunits. The enzyme had a specificity for cellobiose, requiring Pi and Mg2+ for phosphorylation, but not for cellodextrin, gentibiose, laminaribiose, lactose, maltose, kojibiose and sucrose. The enzyme showed low thermostability, an optimum pH of 7.6 and a high stability in the presence of 2-mercaptoethanol or dithiothreitol. The Km values for cellobiose and Pi were 1.25 mM and 0.77 mM respectively. Nojirimycin acted as a powerful pure competitive inhibitor (with respect to cellobiose) of the enzyme (Ki = 45 microM). Addition of thiol-blocking agents to the enzyme caused 56% inhibition at 500 microM-N-ethylmaleimide and 100% at 20 microM-p-chloromercuribenzoate.

scholarly journals Purification and some properties of glyoxylate reductase (NADP+) and its functional location in mitochondria in Euglena gracilis z

1985 ◽  
Vol 227 (1) ◽  
pp. 211-216 ◽  
Author(s):  
A Yokota ◽  
S Haga ◽  
S Kitaoka
Keyword(s):  
Gel Electrophoresis ◽  
Glyoxylate Cycle ◽  
Polyacrylamide Gel Electrophoresis ◽  
Intermembrane Space ◽  
Difference Spectra ◽  
Inner Membrane ◽  
Optimum Ph ◽  
Glycollate Dehydrogenase ◽  
Euglena Gracilis Z ◽  
Glyoxylate Reductase

Euglena mitochondria contain both glyoxylate reductase (NADP+) and glycollate dehydrogenase to constitute the glycollate-glyoxylate cycle [Yokota & Kitaoka (1979) Biochem. J. 184, 189-192]. Euglena glyoxylate reductase (NADP+) was purified and its submitochondrial location was determined in order to elucidate the cycle. The purified glyoxylate reductase was homogeneous on polyacrylamide-gel electrophoresis. Difference spectra of the purified enzyme revealed that the enzyme was a flavin enzyme. The Mr of the enzyme was 82 000. The enzyme was specific for NADPH, with an apparent Km of 3.9 microM, and for glyoxylate, with an apparent Km of 45 microM. It was 30% as active with oxaloacetate as with glyoxylate. NADH and hydroxypyruvate did not support the activity at all. The optimum pH was 6.45. Submitochondrial fractionation of purified mitochondria showed that the enzyme was located in the intermembrane space and loosely bound to the outer surface of the inner membrane. These properties and the submitochondrial localization of NADPH-glyoxylate reductase facilitate the operation of the glycollate-glyoxylate cycle in combination with glycollate dehydrogenase, which is tightly bound to the inner membrane of Euglena mitochondria.

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