Purification and properties of amylase produced by a moderately halophilic Acinetobacter sp.

1978 ◽  
Vol 24 (9) ◽  
pp. 1017-1023 ◽  
Author(s):  
Hiroshi Onishi ◽  
Osamu Hidaka
Keyword(s):  
Gel Electrophoresis ◽  
Enzyme Production ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Soluble Starch ◽  
Moderately Halophilic ◽  
Molecular Weights ◽  
Purification And Properties ◽  
Acinetobacter Sp ◽  
Sea Sands

A moderately halophilic Acinetobacter sp., capable of producing dextrinogenic amylase, was isolated from sea-sands. Maximum enzyme production was obtained when the bacterium was cultivated aerobically in media containing 1 to 2 M NaCl or 1 M KCl. Two kinds of amylase, amylases I and II were purified from the culture filtrate to an electrophoretically homogenous state by glycogen-complex formation, DEAE-Sephadex A-50 chromatography, and Sephadex G-200 gel filtration. Both enzymes had maximal activity at pH 7.0 in 0.2 to 0.6 M NaCl or KCl at 50 to 55 °C. The activities were lost by dialysis against distilled water. Molecular weights for amylases I and II were estimated to be 55 000 and 65 000 respectively by SDS-gel electrophoresis. The action pattern on amylose, soluble starch, and glycogen showed that the products were maltose and maltotriose.

Purification and properties of extracellular endoxylanases from alkalophilic thermophilic Bacillus sp.

1992 ◽  
Vol 38 (5) ◽  
pp. 436-442 ◽  
Author(s):  
Devyani Dey ◽  
Jyoti Hinge ◽  
Abhay Shendye ◽  
Mala Rao
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Bacillus Sp ◽  
Thermophilic Bacillus ◽  
Molecular Weights ◽  
Purification And Properties ◽  
Similar Temperature

An alkalophilic thermophilic Bacillus sp. (NCIM 59) isolated from soil produced two types of cellulase-free xylanase at pH 10 and 50 °C. The two enzymes (xylanase I and II) were purified to homogeneity by ethanol precipitation followed by Bio-Gel P-10 gel filtration and preparative polyacrylamide gel electrophoresis. The molecular weights of xylanase I and II were estimated to be 35 000 and 15 800, respectively, by sodium dodecyl sulfate gel electrophoresis. The enzymes exhibited immunological cross-reactivity and were glycoproteins. They had similar temperature (50–60 °C) and pH (6) optima. Both xylanases were stable at 50 °C at pH 7 for 4 days. However, xylanase I was comparatively more stable than xylanase II at 60 °C. The isoelectric points of xylanase I and II were 4 and 8, respectively. The apparent Km values, using xylan as substrate, were 1.58 and 3.5 mg/mL, and Vmax values were 0.0172 and 0.742 μmol∙min−1∙mg−1, respectively. Both xylanases were inhibited by N-bromosuccinimide, suggesting the involvement of tryptophan in the active site. The hydrolysis patterns demonstrated that the xylanases were endoenzymes. Xylanase I and II yielded mainly xylobiose, xylotriose, and higher xylooligosaccharides, with traces of xylose from xylan. Key words: cellulase-free xylanase, alkalophilic thermophilic Bacillus sp., enzyme purification, characterization.

Purification and properties of mercuric reductase from Yersinia enterocolitica 138A14

1993 ◽  
Vol 39 (2) ◽  
pp. 193-200 ◽  
Author(s):  
Mohamed Blaghen ◽  
Dominique J. M. Vidon ◽  
Mohamed Said El Kebbaj
Keyword(s):  
Molecular Weight ◽  
Yersinia Enterocolitica ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Mercury Resistance ◽  
Thiol Compounds ◽  
Mercuric Reductase ◽  
Purification And Properties ◽  
Layer Chromatography

A mercuric ion-reducing flavoprotein was purified from Yersinia enterocolitica 138A14 using dye matrix affinity chromatography. The purified enzyme had a characteristic absorption spectrum similar to those of flavin compounds, and FAD was detected as a part of the purified enzyme by thin-layer chromatography. Freshly purified preparations of the enzyme showed a single band on SDS polyacrylamide gel electrophoresis with a molecular weight of 70 000. The isolated enzyme had a molecular weight of about 200 000 as determined by gel filtration and disc gel electrophoresis. These results suggest an apparently trimeric structure of the enzyme. Dithiothreitol treatment disrupted the trimer into a dimeric structure of 140 000. Along with ageing, as well as limited proteolytic digestion, the enzyme evolved to give a dimeric molecule of 105 000 composed of two identical subunits of 52 000. The combination of the purified enzyme with HgCl2, or unexpectedly with merthiolate, oxidised the NADPH, which was followed spectrophotometrically. The Km for HgCl2 was dependent on the concentration of exogenous thiol compounds. A comparison of physical properties as well as kinetic characteristics indicated that the enzyme from Y. enterocolitica 138A14 is similar to mercuric reductases isolated from other mercury-resistant bacteria.Key words: Yersinia enterocolitica, mercury resistance, mercuric reductase.

II. Mitt.: Auflösung der Chromoproteidfraktion aus Rattenleber-Ribosomen mit Hilfe der Polyacrylamidgel-Elektrophorese

1970 ◽  
Vol 25 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Wolfram Domschke ◽  
Jürgen G. Meyer-Bertenrath
Keyword(s):  
Gel Electrophoresis ◽  
Ribosomal Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
The Other ◽  
Blue Staining ◽  
Ribosomal Subunits ◽  
Molecular Weights ◽  
The One ◽  
Liver Ribosomes

After preparation of a coloured protein component containing iron from rat liver ribosomes 1 this fraction was submitted to detailed analysis by means of polyacrylamide gel electrophoresis. Thus it may be separated into one main and two secondary bands, which do not contain RNA detectable by methylene blue staining. The ferric content of all bands can be demonstrated by staining with 2,4-dinitroso-1,3-naphthalenediol 2. These bands are to be found in the large ribosomal subunits as well as in the small one in qualitative conformity, they differ, however, in their quantitative relations to each other depending on origin. LiCl-extraction as described for the preparation of ribosomal proteins causes dissociation of the chromoproteid fraction into six bands possessing lower molecular weights each than the original bands.The chromoproteids, localized in the large ribosomal subunits on the one hand and in the small subunits on the other hand were prepared differentiatedly by gel filtration. Results show the chromoproteid represented in the 57-S-subunit on a bigger scale than the nucleoproteid part, on the contrary, the 29-S-subunit is constructed of RNA-containing material preferably.

Studies on Molecular Weights of Two Peptide Hormones from the Urophysis of White Sucker (Catostomus commersoni)

1975 ◽  
Vol 53 (2) ◽  
pp. 242-247 ◽  
Author(s):  
Graham Moore ◽  
Anita Letter ◽  
Maria Tesanovic ◽  
Karl Lederis
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Hypotensive Activity ◽  
White Sucker ◽  
Urotensin Ii ◽  
Catostomus Commersoni ◽  
Molecular Weights ◽  
Long Acting

The molecular weights of two active principles extracted from the urophysis of the teleost fish Catostomus commersoni in 0.1 N HCl or in 0.25% acetic acid have been investigated by gel filtration chromatography and SDS–polyacrylamide gel electrophoresis. Two peptides with urotensin I (long-acting rat hypotensive) activity and two peptides with urotensin II (fish smooth muscle stimulating) activity were found by these procedures. The smaller of the two urotensin I peptides (molecular weight 1200–1700), designated urotensin IS, was shown to be a fragment of the larger peptide (molecular weight 2300–3000) which is produced by acid hydrolysis without loss of rat hypotensive activity. The two urotensin II peptides are suggested to represent either a monomer and a dimer or open and closed forms of a peptide.

scholarly journals Purification and properties of two glyoxylate reductases from a species of Pseudomonas

1966 ◽  
Vol 101 (3) ◽  
pp. 781-791 ◽  
Author(s):  
LN Cartwright ◽  
RP Hullin
Keyword(s):  
Equilibrium Constants ◽  
Maximal Activity ◽  
High Order ◽  
Thiol Groups ◽  
Molecular Weights ◽  
Free Thiol ◽  
Purification And Properties ◽  
Keto Acids ◽  
Michaelis Constants

1. Two enzymes that catalyse the reduction of glyoxylate to glycollate have been separated and purified from a species of Pseudomonas. Their molecular weights were estimated as 180000. 2. Reduced nicotinamide nucleotides act as the hydrogen donators for the enzymes. The NADH-linked enzyme is entirely specific for its coenzyme but the NADPH-linked reductase shows some affinity towards NADH. 3. Both enzymes convert hydroxypyruvate into glycerate. 4. The glyoxylate reductases show maximal activity at pH6.0-6.8, are inhibited by keto acids and are strongly dependent on free thiol groups for activity. 5. The Michaelis constants for glyoxylate and hydroxypyruvate were found to be of a high order. 6. The reversibility of the reaction has been demonstrated for both glyoxylate reductases and the equilibrium constants were determined. 7. The reduction of glyoxylate and hydroxypyruvate is not stimulated by anions.

scholarly journals Purification and properties of α-galactosidases from Vicia faba seeds

1969 ◽  
Vol 113 (1) ◽  
pp. 49-55 ◽  
Author(s):  
P. M. Dey ◽  
J. B. Pridham
Keyword(s):  
Amino Acid ◽  
Vicia Faba ◽  
Aromatic Amino Acid ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Thermal Stabilities ◽  
Molecular Weights ◽  
Purification And Properties ◽  
Enzyme I ◽  
Carbohydrate Contents

Two forms of α-galactosidase, I and II, exist in Vicia faba seeds and these have been purified 3660- and 337-fold respectively. They behaved as homogeneous preparations when examined by ultracentrifugation, disc electrophoresis and gel filtration. The apparent molecular weights of enzymes I and II, as determined by gel filtration, were 209000 and 38000 respectively. The carbohydrate contents of enzymes I and II were 25% and 2·8% respectively, and the enzymes differed in their aromatic amino acid compositions. Enzyme I was split into six inactive subunits in the presence of 6m-urea. α-Galactosidases I and II showed different pH optima and Km and Vmax. values with p-nitrophenyl α-d-galactoside and raffinose as substrates, and also differed in their thermal stabilities.

scholarly journals Two Forms of NAD-Dependent d-Mandelate Dehydrogenase in Enterococcus faecalis IAM 10071

2002 ◽  
Vol 68 (2) ◽  
pp. 947-951 ◽  
Author(s):  
Yusuke Tamura ◽  
Atsuko Ohkubo ◽  
Saho Iwai ◽  
Yoichiro Wada ◽  
Takeshi Shinoda ◽  
...  
Keyword(s):  
High Activity ◽  
Enterococcus Faecalis ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Molecular Weights

ABSTRACT Two forms of NAD-dependent d-mandelate dehydrogenase (d-ManDHs) were purified from Enterococcus faecalis IAM 10071. While these two enzymes consistently exhibited high activity toward large 2-ketoacid substrates that were branched at the C3 or C4 position, they gave distinctly different Km and V max values for these substrates and had distinct molecular weights by gel electrophoresis and gel filtration.

ISOLATION OF LH, FSH AND TSH FROM HUMAN PITUITARIES AFTER THE REMOVAL OF HGH

1974 ◽  
Vol 77 (3) ◽  
pp. 485-497 ◽  
Author(s):  
P. A. Torjesen ◽  
T. Sand ◽  
N. Norman ◽  
O. Trygstad ◽  
I. Foss
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Disc Electrophoresis ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Molecular Weights ◽  
Pituitary Glands

ABSTRACT Highly purified human LH, FSH and TSH were isolated from batches of 300 frozen pituitary glands (200 g) by pH, acetone and ethanol fractionation, Sephadex gel filtration, ion-exchange chromatography on DEAE-cellulose and CM-Sephadex, and preparative polyacrylamide-gel electrophoresis. Sodium dodecyl-sulphate (SDS) polyacrylamide gel electrophoresis was used in order to check the purity, the identity and the molecular weight of the purified LH, FSH and TSH. This procedure showed that the hormone preparations consisted of two subunits with molecular weights of: LH: 21 300 and 17 900, FSH: 22 100 and 18 300 and TSH: 20 800 and 16 400. The purity of the hormone preparations was also evaluated by analytical disc electrophoresis at pH 8.9. The purified hormone preparations had radioimmunological activity as follows: LH: 20 000 IU/mg, FSH: 16 500 IU/mg and TSH: 5 IU/mg. All preparations had high biological potency.

scholarly journals Purification and properties of 3-hexulose phosphate synthase and phospho-3-hexuloisomerase from Methylococcus capsulatus

1974 ◽  
Vol 144 (3) ◽  
pp. 477-486 ◽  
Author(s):  
T Ferenci ◽  
T Strøm ◽  
J R Quayle
Keyword(s):  
Equilibrium Constants ◽  
Gel Filtration ◽  
Reverse Reaction ◽  
Methylococcus Capsulatus ◽  
Forward Reaction ◽  
Bivalent Cation ◽  
Molecular Weights ◽  
Purification And Properties ◽  
Michaelis Constants ◽  
Phosphate Synthase

3-Hexulose phosphate synthase and phospho-3-hexuloisomerase were purified 40- and 150-fold respectively from methane-grown Methylococcus capsulatus. The molecular weights of the enzymes were approximately 310000 and 67000 respectively, as determined by gel filtration. Dissociation of 3-hexulose phosphate synthase into subunits of molecular weight approx. 49000 under conditions of low pH or low ionic strength was observed. Within the range of compounds tested, 3-hexulose phosphate synthase is specific for formaldehyde and d-ribulose 5-phosphate (forward reaction) and d-arabino-3-hexulose 6-phosphate (reverse reaction), and phospho-3-hexuloisomerase is specific for d-arabino-3-hexulose 6-phosphate (forward reaction) and d-fructose 6-phosphate (reverse reaction). A bivalent cation is essential for activity and stability of 3-hexulose phosphate synthase; phospho-3-hexuloisomerase is inhibited by many bivalent cations. The pH optima of the two enzymes are 7.0 and 8.3 respectively and the equilibrium constants are 4.0×10-5m and 1.9×102m respectively. The apparent Michaelis constants for 3-hexulose phosphate synthase are: d-ribulose 5-phosphate, 8.3×10-5m; formaldehyde, 4.9×10-4m; d-arabino-3-hexulose 6-phosphate, 7.5×10-5m. The apparent Michaelis constants for phospho-3-hexuloisomerase are: d-arabino-3-hexulose 6-phosphate, 1.0×10-4m; d-fructose 6-phosphate, 1.1×10-3m.

scholarly journals The purification and properties of the lectin from potato tubers, a hydroxyproline-containing glycoprotein

1973 ◽  
Vol 135 (2) ◽  
pp. 307-314 ◽  
Author(s):  
Anthony K. Allen ◽  
Albert Neuberger
Keyword(s):  
Amino Acid ◽  
Wheat Germ ◽  
Gel Filtration ◽  
Indirect Evidence ◽  
Basic Amino Acid ◽  
Potato Tubers ◽  
Molecular Weights ◽  
Purification And Properties ◽  
Sepharose 4B ◽  
Abundant Amino Acid

1. Potato lectin has been purified and shown to be a glycoprotein containing about 50% of carbohydrate. Most of the sugar residues (92%) are arabinose; small amounts of galactose, glucose and glucosamine are also present. 2. The most abundant amino acid is hydroxyproline (16% of the residues), 11.5% of the residues are half-cystine and phenylalanine is absent. The lectin also contains about one residue/molecule of a basic amino acid, not usually found in proteins, which has been tentatively identified as ornithine. There is indirect evidence that the components of the glycoprotein are linked through hydroxyproline and arabinose. 3. By gel filtration in 6m-guanidine–HCl on Sepharose 4B, it was found that both the native glycoprotein and its S-carboxymethylated derivative had subunit molecular weights of 46000 (±5000). In a non-denaturing solution, two of these units appear to be associated. 4. The lectin is specifically inhibited in its agglutination reaction by oligosaccharides that contain N-acetylglucosamine. Its specificity is similar to, but not identical with, that of wheat-germ agglutinin.

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