Two types of xylanases of alkalophilic Bacillus sp. No. C-125

1985 ◽  
Vol 31 (6) ◽  
pp. 538-542 ◽  
Author(s):  
H. Honda ◽  
T. Kudo ◽  
Y. Ikura ◽  
K. Horikoshi
Keyword(s):  
Ammonium Sulfate ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Bacillus Sp ◽  
Ammonium Sulfate Precipitation ◽  
Molecular Weights ◽  
Protein Molecules ◽  
Activity Curve ◽  
Alkalophilic Bacillus ◽  
Hydrolysis Of

One alkalophilic Bacillus sp. strain C-125 (FERM No. 7344) was isolated from soil. From this organism, two types of xylanases, designated xylanase A and xylanase N, were purified by an ammonium sulfate precipitation followed by Biogel P-30 gel filtration, DEAE-cellulose chromatography, and Sephadex G-75 gel filtration. The molecular weights of xylanase A and N were estimated as 43 000 and 16 000, respectively. Immunological experiments indicated that xylanase A and xylanase N were entirely different protein molecules. Xylanase N was most active at pH 6.0–7.0, but xylanase A had a very broad pH activity curve (pH 6–10) and was still active even at pH 12.0. The maximum hydrolysis of xylan by the enzymes was about 25%. Both enzymes split xylan and yielded xylobiose and higher oligosaccharides but could hydrolyze neither xylobiose nor xylotriose. Trans xylosidation activities were detected in both enzymes.

Extracellular proteolytic enzymes of psychrophilic bacteria. I. Purification and some properties of enzymes of an obligately psychrophilic red-pigmented bacterium

1968 ◽  
Vol 14 (3) ◽  
pp. 215-224 ◽  
Author(s):  
Y. Nunokawa ◽  
I. J. McDonald
Keyword(s):  
Metal Ions ◽  
Ammonium Sulfate ◽  
Synthetic Peptides ◽  
Proteolytic Enzymes ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Psychrophilic Bacterium ◽  
Psychrophilic Bacteria ◽  
Fraction I ◽  
Hydrolysis Of

Proteinase in culture fluids of an obligately psychrophilic bacterium was precipitated by ammonium sulfate and fractionated by gel filtration and DEAE-cellulose chromatography. Three purified fractions (I-1, I-2, and III-1) with proteinase activity were obtained. On the basis of reactions and characteristics (i.e. effect of pH, heat, and metal ions on activity and stability, hydrolysis of synthetic peptides and of natural proteins) fractions I-1 and III-1 appeared to be very similar whereas fraction I-2 was different. When proteinase preparations were examined by electrophoresis, fractions I-1 and III-1 gave similar patterns; fraction I-2 gave a different one. From the results it is suggested that the organism produces two proteinases and that possibly fraction I-1 represents an aggregation of molecules of III-1 and that fraction I-2 is a different proteinase.

scholarly journals Extraction and Partial Purification of Mouse Uterine Alkaline Phosphatase

1979 ◽  
Vol 32 (2) ◽  
pp. 153 ◽  
Author(s):  
RN Murdoch ◽  
DJ Kay ◽  
WJ Capper
Keyword(s):  
Alkaline Phosphatase ◽  
Ammonium Sulfate ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Ammonium Sulfate Precipitation ◽  
Pregnant Mice ◽  
Triton X

Alkaline phosphatase in uterine homogenates from day 7 pregnant mice was solubilized using 0�2 % (v/v) Triton X-100 and extracted with 20% (v/v) n-butanol. The procedure, which resulted in 182- fold purification, included ammonium sulfate precipitation, DEAE-cellulose anion exchange chromatography and Sephadex 0200 gel filtration.

scholarly journals α-Crystallin. The isolation and characterization of distinct macromolecular fractions

1971 ◽  
Vol 124 (2) ◽  
pp. 337-343 ◽  
Author(s):  
Abraham Spector ◽  
Lu-Ku Li ◽  
Robert C. Augusteyn ◽  
Arthur Schneider ◽  
Thomas Freund
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Chemical Difference ◽  
Molecular Weights ◽  
Isolation And Characterization ◽  
Different Populations ◽  
Molecular Weight Fractions

α-Crystallin was isolated from calf lens periphery by chromatography on DEAE-cellulose and gel filtration. Three distinct populations of macromolecules have been isolated with molecular weights in the ranges approx. 6×105−9×105, 0.9×106−4×106and greater than 10×106. The concentration of macromolecules at the molecular-weight limits of a population are very low. The members of the different populations do not appear to be in equilibrium with each other. Further, in those molecular-weight fractions investigated, no equilibrium between members of the same population was observed. The population of lowest molecular weight comprises 65–75% of the total material. The amino acid and subunit composition of the different-sized fractions appear very similar, if not identical. The only chemical difference observed between the fractions is the presence of significant amounts of sugar in the higher-molecular-weight fractions. Subunit molecular weights of approx. 19.5×103and 22.5×103were observed for all α-crystallin fractions.

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

scholarly journals Anti-coagulant Activity of Isolated and Partially Characterized Proteoglycans and Glycosaminoglycans from African Night Crawler (Eudrilus eugeniae Kinberg)

KIMIKA ◽  
2015 ◽  
Vol 26 (2) ◽  
pp. 31-38
Author(s):  
Mia Clare Marie L. Bercansil ◽  
Miko Lorenzo J. Belgado
Keyword(s):  
Hyaluronic Acid ◽  
Infrared Spectroscopy ◽  
Enzymatic Hydrolysis ◽  
Gel Filtration ◽  
Acetate Buffer ◽  
Eudrilus Eugeniae ◽  
Gel Filtration Chromatography ◽  
Molecular Weights ◽  
Coagulant Activity ◽  
Hydrolysis Of

Proteoglycans and glycosaminoglycans were isolated from African night crawler (Eudrilus eugeniae Kinberg) and partially characterized proteoglycans (3.04 % of lyophilized worm) were liberated from the defatted and depurinated worm samples by dissociative method using 4M urea in acetate buffer. Glycosaminoglycans (12.47% of proteoglycan extract) were extracted using enzymatic hydrolysis of the proteoglycan extract with papain. Gel filtration chromatography using Sepharose CL-4B was used to purify and estimate the molecular weights of the proteoglycan and glycosaminoglycan fractions. Three proteoglycan fractions PGF1, PGF2 and PGF3 with estimated molecular weigths 860 kDa, 181 kDa and 3 kDa, respectively were identified as monitored by the Bradford and modified carbazole assay. Two glycosaminoglycan fractions - GF1 (MW = 860 kDa) and GF2 (MW=140 kDa) were identified using the modified carbazole assay. Infrared spectroscopy of the GF1 and GF2 showed the possible identities of the fractions. GF1 may be a hyaluronic acid and GF2 is possibly chondroitin. Anti-coagulant assay for the extracts and fractions revealed that the glycosaminoglycan isolate has anti-coagulant activity but not the GF1 and GF2 fractions individually.

scholarly journals Purification and characterization of ascamycin-hydrolysing aminopeptidase from Xanthomonas citri

1986 ◽  
Vol 233 (2) ◽  
pp. 459-463 ◽  
Author(s):  
H Osada ◽  
K Isono
Keyword(s):  
Antibacterial Activity ◽  
Enzyme Inhibitors ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Homogeneous State ◽  
Xanthomonas Citri ◽  
Gram Positive Bacteria ◽  
Xanthomonas Species ◽  
Hydrolysis Of

A nucleoside antibiotic, ascamycin (9-beta-[5′-0-(N-L-alanyl) sulphamoyl-D-ribofuranosyl]-2-chloroadenine), has a selective antibacterial activity against Xanthomonas species. When ascamycin was dealanylated, dealanylascamycin showed a broad antibacterial activity against various Gram-negative and Gram-positive bacteria. Xanthomonas citri is susceptible to ascamycin by virtue of the ascamycin-dealanylating enzyme on the cell surface [Osada & Isono (1985) Antimicrob. Agents Chemother. 27, 230-233]. The enzyme (Xc aminopeptidase) was purified from X. citri cells by successive DEAE-cellulose, chromatofocusing and Sephadex G-100 column chromatography to a homogeneous state. The purified enzyme exhibited a single band with an Mr of 38 000 in SDS/polyacrylamide-gel electrophoresis. Gel filtration on a calibrated column indicated a similar Mr value. The isoelectric point of the enzyme was 5.7. The enzyme catalysed the hydrolysis of the alanyl group of ascamycin and liberated alanine from the sulphamoyl nucleoside. The enzyme also catalysed the hydrolysis of L-proline beta-naphthylamide and L-alanine beta-naphthylamide. The optimal pH and temperature for enzyme activity were pH 7.5-8.0 and 35-40 degrees C respectively. The enzyme was inhibited by thiol-enzyme inhibitors (i.e. rho-chloromercuribenzoate and N-ethylmaleimide), but was not affected by various naturally occurring aminopeptidase inhibitors (i.e. amastatin, bestatin, pepstatin and leupeptin). Mn2+ and Mg2+ activated the enzyme, whereas Cu2+, Zn2+ and Cd2+ were inhibitory.

scholarly journals Purification and characterization of two human erythrocyte arylamidases preferentially hydrolysing N-terminal arginine or lysine residues

1978 ◽  
Vol 175 (3) ◽  
pp. 1051-1067 ◽  
Author(s):  
K K Mäkinen ◽  
P L Mäkinen
Keyword(s):  
Substrate Inhibition ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Gel Permeation Chromatography ◽  
Ph Optimum ◽  
Preparative Scale ◽  
Molecular Weights ◽  
Gel Permeation ◽  
Lysine Residues ◽  
Enzyme I

Two arylamidases (I and II) were purified from human erythrocytes by a procedure that comprised removal of haemoglobin from disrupted cells with CM-Sephadex D-50, followed by treatment of the haemoglobin-free preparation subsequently with DEAE-cellulose, gel-permeation chromatography on Sephadex G-200, gradient solubilization on Celite, isoelectric focusing in a pH gradient from 4 to 6, gel-permeation chromatography on Sephadex G-100 (superfine), and finally affinity chromatography on Sepharose 4B covalently coupled to L-arginine. In preparative-scale purifications, enzymes I and II were separated at the second gel-permeation chromatography. Enzyme II was obtained as a homogeneous protein, as shown by several criteria. Enzyme I hydrolysed, with decreasing rates, the L-amino acid 2-naphtylamides of lysine, arginine, alanine, methionine, phenylalanine and leucine, and the reactions were slightly inhibited by 0.2 M-NaCl. Enzyme II hydrolysed most rapidly the corresponding derivatives of arginine, leucine, valine, methionine, proline and alanine, in that order, and the hydrolyses were strongly dependent on Cl-. The hydrolysis of these substrates proceeded rapidly at physiological Cl- concentration (0.15 M). The molecular weights (by gel filtration) of enzymes I and II were 85 000 and 52 500 respectively. The pH optimum was approx. 7.2 for both enzymes. The isoelectric point of enzyme II was approx. 4.8. Enzyme I was activated by Co2+, which did not affect enzyme II to any noticeable extent. The kinetics of reactions catalysed by enzyme I were characterized by strong substrate inhibition, but enzyme II was not inhibited by high substrate concentrations. The Cl- activated enzyme II also showed endopeptidase activity in hydrolysing bradykinin.

scholarly journals Isolation of palmitoyl-CoA hydrolases from human blood platelets

1981 ◽  
Vol 199 (3) ◽  
pp. 639-647 ◽  
Author(s):  
R K Berge ◽  
L E Hagen ◽  
M Farstad
Keyword(s):  
Human Blood ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Cytosol Fraction ◽  
Molecular Weights ◽  
Apparent Homogeneity ◽  
Coa Hydrolase ◽  
Human Blood Platelets

The palmitoyl-CoA hydrolase activity, which in human blood platelets is mainly localized in the cytosol fraction [Berge, Vollset & Farstad (1980) Scand. J. Clin. Lab. Invest. 40, 271--279], was found to be extremely labile. Inclusion of glycerol or palmitoyl-CoA stabilized the activity during preparation. Gel-filtration studies revealed multiple forms of the enzyme with molecular weights corresponding to about 70 000, 40 000 and 24 000. The relative recovery of the mol.wt.-70 000 form was increased by the presence of 20% (v/v) glycerol or 10 microM-palmitoyl-CoA. The three enzyme forms are probably unrelated, since they were not interconvertible. The three different species of palmitoyl-CoA hydrolase were purified by DEAE-cellulose and hydroxyapatite chromatography, isoelectric focusing and high-pressure liquid chromatography (h.p.l.c.) to apparent homogeneity. The three enzymes had isoelectric points (pI) of 7.0, 6.1 and 4.9. The corresponding molecular weights were 27 000--33 000, 66 000--72 000 and 45 000--49 000, calculated from h.p.l.c. and Ultrogel AcA-44 chromatography. The apparently purified enzymes were unstable, as most of the activity was lost during purification. The enzyme with an apparent molecular weight of 45 000--49 000 was split into fractions with molecular weights of less than 10 000 by re-chromatography on h.p.l.c. concomitantly with a loss of activity. The stimulation of the activity by the presence of serum albumin seems to depend on the availability of palmitoyl-CoA, as has been reported for other palmitoyl-CoA hydrolases. [Berge & Farstad (1979) Eur. J. Biochem. 96, 393--401].

scholarly journals Purification and Characterization ofα -l-Arabinopyranosidase andα -l-Arabinofuranosidase from Bifidobacterium breve K-110, a Human Intestinal Anaerobic Bacterium Metabolizing Ginsenoside Rb2 and Rc

2003 ◽  
Vol 69 (12) ◽  
pp. 7116-7123 ◽  
Author(s):  
Ho-Young Shin ◽  
Sun-Young Park ◽  
Jong Hwan Sung ◽  
Dong-Hyun Kim
Keyword(s):  
Ammonium Sulfate ◽  
Column Chromatography ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Bifidobacterium Breve ◽  
Ammonium Sulfate Fractionation ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Ginsenoside Rb2

ABSTRACT Two arabinosidases, α-l-arabinopyranosidase (no EC number) and α-l-arabinofuranosidase (EC 3.2.1.55), were purified from ginsenoside-metabolizing Bifidobacterium breve K-110, which was isolated from human intestinal microflora. α-l-Arabinopyranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, QAE-cellulose, and Sephacryl S-300 HR column chromatography, with a final specific activity of 8.81 μmol/min/mg.α -l-Arabinofuranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, Q-Sepharose, and Sephacryl S-300 column chromatography, with a final specific activity of 6.46 μmol/min/mg. The molecular mass ofα -l-arabinopyranosidase was found to be 310 kDa by gel filtration, consisting of four identical subunits (77 kDa each, measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]), and that ofα -l-arabinofuranosidase was found to be 60 kDa by gel filtration and SDS-PAGE. α-l-Arabinopyranosidase and α-l-arabinofuranosidase showed optimal activity at pH 5.5 to 6.0 and 40°C and pH 4.5 and 45°C, respectively. Both purified enzymes were potently inhibited by Cu2+ and p-chlormercuryphenylsulfonic acid.α -l-Arabinopyranosidase acted to the greatest extent on p-nitrophenyl-α-l-arabinopyranoside, followed by ginsenoside Rb2. α-l-Arabinofuranosidase acted to the greatest extent on p-nitrophenyl-α-l-arabinofuranoside, followed by ginsenoside Rc. Neither enzyme acted on p-nitrophenyl-β-galactopyranoside or p-nitrophenyl-β-d-fucopyranoside. These findings suggest that the biochemical properties and substrate specificities of these purified enzymes are different from those of previously purified α-l-arabinosidases. This is the first reported purification ofα -l-arabinopyranosidase from an anaerobic Bifidobacterium sp.

scholarly journals PURIFICATION AND CHARACTERIZATION OF SALIVARY AMYLASE INHIBITOR EXTRACTED FROM BARLEY

2016 ◽  
Vol 47 (4) ◽  
Author(s):  
Abood & Hakeem
Keyword(s):  
Ammonium Sulfate ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Alpha Amylase ◽  
Ionic Exchange ◽  
Inhibition Activity ◽  
Amylase Inhibitors

Amylase inhibitors were purified by many sequential steps included concentration by gradual addition of ammonium sulfate at  saturation ratios. ranged from 0 to 90% . The best ratio of saturation was found to be 70% as the specific activity and inhibition activity toward Human alpha-amylase(HAS)  were the highest ( 8 U/mg and 6 U/ml respectively as compared to those of the rest ratios, the ratio of saturation with ammonium sulfate 60 % and then 50%, (5.8 ,5.5  )U/ml and( 7.7 ،7 )U/mg respectively for inhibition activity and specific activity and for  40% ,30%20%  saturation  the inhibition activity and specific activity were(5 ،4.8 ،4 ) u/ml (6.6 ،6 ،5.8) u/mg respectively .The precepitation step was followed by ionic exchange chromatography technique by DEAE-cellulose column( 3×11 )cm and the results showed that there was one peak with inhibition activity toward (HAS). Further  purification steps were conducted using gel filtration on Sephacryl S-200 column    (1.5  ×  60)cm; the purification folds was5.59 times with outcome of 46.5%.The results of alpha-amylase inhibitors characterization showed that the molecular weight was about 23.44 and 22.9  kDa  as determined by electrophoresis and gel filteration respectively.                                         

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