scholarly journals  α-Glucosidase and β-glucosidase from psychrotrophic strain arthrobacter sp. C2-2

2011 ◽  
Vol 23 (No. 3) ◽  
pp. 116-120 ◽  
Author(s):  
E. Benešová ◽  
M. Marková ◽  
B. Králová
Keyword(s):  
Exchange Chromatography ◽  
Gel Chromatography ◽  
Bacterial Strains ◽  
Molecular Weights ◽  
Ammonium Sulphate Precipitation ◽  
Donor And Acceptor ◽  
Alpha Galactosidase ◽  
Beta Glucosidase ◽  
Psychrotrophic Strain ◽  
Alpha Glucosidase

In this work six psychophilic and psychrotrophic bacterial strains were screened for the presence of different glycosidase activities (&alpha;-galactosidase, &alpha;-glucosidase, &beta;-glucosidase, &alpha;-mannosidase and &beta;-glucuronidase). Nine enzymes were found and their elementary characteristics were measured (t<sub>optimum</sub>, pH<sub>optimum</sub>, K<sub>m</sub>, V<sub>lim</sub>).Two enzymes with the highest activities at low temperatures were chosen for the next study, i.e. &alpha;-glucosidase and &beta;-glucosidase from the psychrotrophic strain Arthrobacter sp. C2-2. These enzymes were purified by ammonium sulphate precipitation, by chromatography with hydrophobic interaction, and by ion-exchange chromatography. Their molecular weights (&alpha;-glucosidase &ndash; 76 kDa, &beta;-glucosidase &ndash; 93 kDa) were determined by gel chromatography. In addition to this, it was verified that both of these enzymes are able to catalyse the transglycosylation reaction with the saccharidic donor and acceptor.

PURIFICATION AND CHARACTERISATION OF FIBRINOLYTIC ENZYMES FROM ENDOPHYTIC FUNGI AND LIGNOSUS RHINOCERUS

2016 ◽  
Vol 78 (6-5) ◽  
Author(s):  
Zainon Mohd Noor ◽  
Mohd Sidek Ahmad ◽  
Zaidah Zainal Ariffin
Keyword(s):  
Ion Exchange ◽  
Endophytic Fungi ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Fibrinolytic Enzymes ◽  
Molecular Weights ◽  
Ammonium Sulphate Precipitation ◽  
Fusarium Sp ◽  
Different Sources

Three enzymes FH3, S13 and LR1 from three different sources showed fibrinolytic activities. Two were from endophytic fungal cultures and one from the sclerotium of Lignosus rhinocerus mushroom (LR1). FH3, S13 cultures and LR1, the crude extract of the sclerotium were concentrated and purified by ammonium sulphate precipitation, ion-exchange chromatography and gel-filtration. The molecular weights of the FH3, S13 and LR1 purified enzymes were estimated to be approximately 34kDa, 34kDa and 10kDa, respectively. Maximum fibrinolytic activities were observed for FH3 at pH 7 and 30°C, S13 at pH 8 and 40°C and LR1 at pH 6 and 40°C.  In our earlier paper we identified FH3 as Fusarium sp. and S13 as Penicilium citrinum. 

scholarly journals Subcellular distribution of glycosidases in human polymorphonuclear leucocytes

1978 ◽  
Vol 174 (1) ◽  
pp. 53-59 ◽  
Author(s):  
R F Rest ◽  
M H Cooney ◽  
J K Spitznagel
Keyword(s):  
Subcellular Distribution ◽  
Polymorphonuclear Leucocytes ◽  
Density Gradients ◽  
Glucosidase Activity ◽  
Mitochondrial Fractions ◽  
Specific Granules ◽  
Azurophil Granule ◽  
Alpha Galactosidase ◽  
Beta Glucosidase ◽  
Alpha Glucosidase

The subcellular distribution of nine glycosidases were studied in fractions of homogenized human polymorphonuclear leucocytes (neutrophils) obtained by isopycnic centrifugation through linear sucrose density gradients. The substrates were 4-methylumbelliferyl glycosides. All nine glycosides were hydrolysed by enzymes in neutrophil cytosol fractions, and by enzymes in at least one granule population. alpha-Glucosidase activity sedimented in sucrose density gradients to a point (p = 1.180 g/ml) just above the specific granules, possibly the ‘tertiary’ granule population. The peak corresponding to alpha-glucosidase did not co-sediment with, but considerably overlapped, the peak corresponding to lactoferrin, a marker for specific granules (p = 1.187 g/ml). alpha-Galactosidase activity was found primarily in heavy azurophil granules (p = 1.222 g/ml). alpha-Mannosidase activity was found primarily in light azurophil granules (p = 1.206 g/ml), following the distribution of myeloperoxidase, the commonly used azurophil granule marker. beta-Glucosidase activity was concentrated in mitochondrial fractions (p = 1.160 g/ml). All other glycosidases presented complex distributions, with activities not restricted to one granule class. Granule-associated glycosidase activities were increased 2–38 times when measured in the presence of 0.05% Triton X-100, indicating latency of the enzymes within granules.

scholarly journals Purification and Partial Characterization of Rat Fibrinogen (rat-Fbg)

1977 ◽  
Author(s):  
W. Nieuwenhuizen ◽  
Irina A.M. van Ruijven-Vermeer
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Proteolytic Enzymes ◽  
Mammalian Species ◽  
Rat Plasma ◽  
Gel Chromatography ◽  
Molecular Weights ◽  
Ammonium Sulphate Precipitation ◽  
Proteolytic Activities

Rat-Fbg was purified from rat plasma using Sepharose-1ysi ne chromatography, repeated ammonium sulphate precipitation (35% saturation) and gel chromatography on Sepharose 6B.In order to minimize proteolytic activity, rats were injected intravenously with Trasylol before bleeding, and the collected blood was treated with Trasylol and DFP.A preparation was obtained, which was 95% clottable and showed a single band on SDS-poly-acrylamide gel electrophoresis. Alanine was the only detectable am i no-term i na 1 amino acid.After reduction and modification of the SH groups the material could be separated into three distinct chains (Aα, Bβ and γ) by pore-limit Polyacrylamide slab-gel electrophoresis in sod i urn dodecyl sulphate. The amino acid composition of the whole Fbg and of the separated modified chains were determined. The molecular weights were 61,000, 58,000 and 51,000 for Aα, Bβ and γ-chains, respectively.In as far as the chains are concerned, our results are in contrast with the findings of Bouma et al. (J. Biol. Chem. 250(1975) 4678), who could not discriminate between Aα- and Bβ-chains in SDS-polyacry1 am i de gel electrophoresis. Evidence will be presented that this can be due to Aa-chain degradation caused by incomplete inhibition of proteolytic enzymes during the purification.It is concluded, that complete inhibition of proteolytic activities in all purification steps is essential to obtain native fibrinogen. Moreover, in contrast to the conclusions of Bouma rat-Fbg does not differ essentially from Fbg from other mammalian species.

scholarly journals Functional lysosomal hydrolase size as determined by radiation inactivation analysis

1985 ◽  
Vol 226 (1) ◽  
pp. 283-288 ◽  
Author(s):  
G Dawson ◽  
J C Ellory
Keyword(s):  
Target Size ◽  
Lysosomal Hydrolase ◽  
Lysosomal Hydrolases ◽  
Range Analysis ◽  
Freeze Dried ◽  
Arylsulphatase A ◽  
Alpha Galactosidase ◽  
Beta Glucosidase ◽  
Alpha Glucosidase ◽  
Beta Galactosidase

Electron inactivation analysis with 16 MeV electrons was used to determine the functional target size of a number of commonly studied lysosomal hydrolases. Observed values ranged from a low of 62 000 +/- 4000 Da for beta-galactosidase to a high of 200 000 +/- 17 500 Da (mouse beta-glucuronidase). One group of lysosomal hydrolases (N-acetyl-beta-glucosaminidase, N-acetyl-beta-galactosaminidase, alpha-galactosidase, beta-mannosidase, beta-glucosidase, arylsulphatase A and sphingomyelinase) had target sizes in the range 100 000-120 000 Da, whereas alpha-glucosidase and alpha-fucosidase exist as complex multimers in the 150 000-160 000 Da range. Analysis of freeze-dried cell material showed little evidence of species (mouse versus human) variation in the functional size of most lysosomal hydrolases with the exception of beta-glucuronidase. Our findings suggest the potential usefulness of lysosomal hydrolases as endogenous marker enzymes in studies where the target size of proteins of unknown molecular mass is to be determined.

scholarly journals Endo-beta-mannanase from the endosperm of seeds of Sesbania virgata (Cav.) Pers. (Leguminosae): purification, characterisation and its dual role in germination and early seedling growth

2006 ◽  
Vol 18 (2) ◽  
pp. 269-280 ◽  
Author(s):  
César Gustavo Serafim Lisboa ◽  
Patrícia Pinho Tonini ◽  
Marco Aurélio Silva Tiné ◽  
Marcos Silveira Buckeridge
Keyword(s):  
Exchange Chromatography ◽  
Gel Chromatography ◽  
Cell Wall Polysaccharides ◽  
Endosperm Tissue ◽  
Ph Optimum ◽  
Radicle Protrusion ◽  
Sesbania Virgata ◽  
Early Seedling ◽  
Mannanase Activity ◽  
Alpha Galactosidase

Galactomannans are storage cell wall polysaccharides present in seeds of some legumes. Their degradation is carried out by three hydrolases (alpha-galactosidase (EC 3.2.1.22), endo-beta-mannanase (EC 3.2.1.78) and ß-mannosidase (EC 3.2.1.25)). In the present study we purified and characterised an endo-beta-mannanase from seeds of Sesbania virgata and addressed its role in germination and seedling development. The polypeptide purified by Ion Exchange Chromatography and Affinity Chromatography on Sepharose-Concanavalin A, showed a pH optimum between 3.5 and 5 at 45ºC and high stability at pH 7.8. The low stability at pH 5 appears to be associated with isoelectric precipitation, in view of the pI of the enzyme being 4.5. The purified enzyme is a glycoprotein with a molecular mass of 26 KDa by SDS-PAGE and 36 KDa by gel chromatography. The purified polypeptide attacked galactomannan from different sources, being more effective on polymers with a lower degree of galactosylation (from carob gum), in comparison with medium or highly galactosylated galactomannans (from guar, S. virgata and fenugreek), respectively. A peak of endo-beta-mannanase activity was detected during radicle protrusion in the endosperm tissue surrounding the radicle and later on in the lateral endosperm. This second peak was associated with the period of reserve mobilisation. Using an antibody raised against coffee endo-beta-mannanase, the enzyme could be detected in immunodot-blots performed with extracts of S. virgata endosperms. The results are consistent with the hypothesis that the peak of endo-mannanase during germination facilitates radicle protrusion through the surrounding endosperm by weakening it in the region close to the radicle tip.

scholarly journals Soil hydrolase activities and kinetic properties as affected by wheat cropping systems of Northeastern China

2010 ◽  
Vol 56 (No. 11) ◽  
pp. 526-532 ◽  
Author(s):  
Y.L. Zhang ◽  
L.J. Chen ◽  
C.X. Sun ◽  
Z.J. Wu ◽  
Z.H. Chen ◽  
...  
Keyword(s):  
Cropping Systems ◽  
Reaction Rates ◽  
Total Carbon ◽  
Kinetic Properties ◽  
Total N ◽  
Alpha Galactosidase ◽  
Beta Glucosidase ◽  
Alpha Glucosidase ◽  
Beta Galactosidase ◽  
Sequential Cropping

Agricultural practices that reduce soil degradation and improve agriculture sustainability are important particularly for dry hilly land of Chaoyang County in the Liaoning Province, North-east China, where cinnamon soils are widely distributed and mainly for wheat production. The impacts of 10-year cropping systems (wheat-cabbage sequential cropping, wheat-corn intercrop, wheat-sunflower rotation, wheat-soybean rotation) on soil enzyme properties of surface-soil (0&ndash;20 cm) were studied. Total carbon, nitrogen, phosphorus and sulfur, and nine soil hydrolases related to nutrient availabilities (&beta;-galactosidase, &alpha;-galactosidase, &beta;-glucosidase, &alpha;-glucosidase, urease, protease, phosphomonoesterase, phosphodiesterase, arylsulphatase) and five enzymes kinetic characters were examined. Wheat-corn intercrop systems had higher total C, total N, total P and total S concentrations than wheat-soybean and wheat-sunflower rotation systems. Most test enzyme activities (&alpha;-galactosidase, &beta;-galactosidase, &alpha;-glucosidase, &beta;-glucosidase, urease, protease, phosphomonoesterase and arylsulphatase) showed the highest activities under wheat-corn intercropping system. Urease, protease and phosphodiesterase activities of wheat-cabbage sequential cropping system were significantly higher than two rotation systems. The maximum reaction rates of enzymes (V<sub>max</sub>) were higher than apparent enzyme activity, which suggests larger potential activity of enzymes, while not all kinetic parameters were adaptive as soil quality indicators in dry hilly cinnamon soil.

scholarly journals Acid hydrolases in leukocytes and platelets of normal subjects and in patients with Gaucher's and Fabry's disease.

1976 ◽  
Vol 143 (4) ◽  
pp. 975-980 ◽  
Author(s):  
E Beutler ◽  
W Kuhl ◽  
F Matsumoto ◽  
G Pangalis
Keyword(s):  
Acid Phosphatase ◽  
Normal Subjects ◽  
Acid Hydrolases ◽  
Gaucher's Disease ◽  
Gaucher’S Disease ◽  
Fabry’S Disease ◽  
Fabry's Disease ◽  
Alpha Galactosidase ◽  
Beta Glucosidase ◽  
Alpha Glucosidase

Lymphocytes, monocytes, neutrophilic granulocytes and platelets were each separated to greater than 95% purity from six normal subjects, three patients with Gaucher's disease, two heterozygotes for Gaucher's disease, and one patient with Fabry's disease. Activities of the following acid hydrolases were determined: "acid" (pH 4.0) beta-glucosidase, pH 5.0 beta-glucosidase, alpha-galactosidase, alpha-arabinosidase, alpha-mannosidase, alpha-glucosidase, beta-glucuronidase, beta-galactosidase, beta-hexosaminidase, and acid phosphatase. Enzymatic activity varied greatly with cell type and the enzyme being measured; the importance of assaying pure preparations especially for heterozygote detection is emphasized. Gaucher's disease patients' cells were found to be deficient in the pH 4.0 acid beta-glucosidase, variable in the pH 5.0 beta-glucosidase, and normal in all other acid hydrolases tested, including acid phosphatase, the activity of which is known to be elevated in plasma. Blood cells of a patient with Fabry's disease were deficient in alpha-galactosidase and normal in all other acid hydrolases tested.

Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

1982 ◽  
Vol 47 (01) ◽  
pp. 014-018 ◽  
Author(s):  
H Sumi ◽  
N Toki ◽  
S Takasugi ◽  
S Maehara ◽  
M Maruyama ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Urinary Trypsin Inhibitor ◽  
Plasmin Activity ◽  
Molecular Weights ◽  
Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

scholarly journals The apparent molecular weights of human intestinal aminopeptidase, enterokinase and maltase in native duodenal fluid

1977 ◽  
Vol 165 (3) ◽  
pp. 583-585 ◽  
Author(s):  
A I Magee ◽  
D A W Grant ◽  
J Hermon-Taylor
Keyword(s):  
Molecular Form ◽  
Gel Chromatography ◽  
Duodenal Fluid ◽  
Molecular Weights ◽  
Induced Changes

The apparent molecular weights of human intestinal aminopeptidase, enterokinase and maltase in native duodenal fluid were estimated by gel chromatography on Sephadex G-200 under different conditions of operational buffer and temperature. No evidence for environmentally induced changes in molecular form was found.

Isolation of specific protease inhibitors from Neurospora crassa

1976 ◽  
Vol 54 (8) ◽  
pp. 699-703 ◽  
Author(s):  
Peter H. Yu ◽  
Maria R. Kula ◽  
Hsin Tsai
Keyword(s):  
Neurospora Crassa ◽  
Protease Inhibitors ◽  
Gel Filtration ◽  
Physiological Role ◽  
Exchange Chromatography ◽  
Proteinase K ◽  
Tryptophan Synthase ◽  
Molecular Weights ◽  
Specific Protease

Four natural protease inhibitors have been partially purified by heat treatment, ion-exchange chromatography and gel filtration from Neurospora crassa. The inhibitory activity has been estimated by measuring the inhibition of proteolysis of casein as well as by the protection of Neurospora tryptophan synthase from proteolytic inactivation. The inhibitors are all oligopeptides and possess molecular weights in the range 5000 – 24 000 and appear to be very specific to Neurospora proteases. They may be classified into two types. The first are specific to Neurospora alkaline protease and the second to acidic protease. None of them exhibited any effect on other proteases including trypsin, chymotrypsin, papain, pepsin, thermolysin, subtilisin and proteinase K. The possible physiological role of these inhibitors is discussed.

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