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 an antibacterial protein from dried fruiting bodies of the wild mushroom Clitocybe sinopica.

2010 ◽  
Vol 57 (1) ◽  
Author(s):  
Suyue Zheng ◽  
Qinghong Liu ◽  
Guoqing Zhang ◽  
Hexiang Wang ◽  
Tzi Bun Ng
Keyword(s):  
Antibacterial Activity ◽  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Fruiting Bodies ◽  
Wild Mushroom ◽  
Antibacterial Protein

A novel antibacterial protein with a molecular mass of 44 kDa has been isolated from dried fruiting bodies of the wild mushroom Clitocybe sinopica. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis showed that the protein was composed of two subunits each with a molecular mass of 22 kDa. Its N-terminal amino-acid sequence, SVQATVNGDKML, has not been reported for other antimicrobial proteins. The purification protocol included ion exchange chromatography on DEAE-cellulose, CM-cellulose and Q-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The antibacterial protein was adsorbed on all three ion exchangers. The antimicrobial activity profile of the protein against tested bacterial and fungal strains disclosed that it possessed potent antibacterial activity against Agrobacterium rhizogenes, A. tumefaciens, A. vitis, Xanthomonas oryzae and X. malvacearum with a minimum inhibitory concentration mostly below 0.6 microM. However, it had no antibacterial activity against Pseudomonas batatae, Erwinia herbicola, Escherichia coli, and Staphylococcus aureus, and no antifungal activity against Setosphaeria turcica, Fusarium oxysporum, Verticillium dahliae, Bipolaris maydis, and B. sativum. The antibacterial antivity against A. tumefaciens was stable after exposure to 20-60 degrees C for 30 min and to pH 4-9 for 1 h.

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 ).

Biosynthesis of glucosyl monophosphoryl undecaprenol and its role in lipoteichoic acid biosynthesis

1982 ◽  
Vol 152 (2) ◽  
pp. 616-625
Author(s):  
D J Mancuso ◽  
T H Chiu
Keyword(s):  
Thin Layer ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Lipoteichoic Acid ◽  
Molar Ratio ◽  
Streptococcus Sanguis ◽  
Chromatographic Profile ◽  
Hydrolysis Of ◽  
Cellulose Column

A glucophospholipid was detected in an incubation mixture containing UDP-glucose, MgCl2, ATP, and a particulate enzyme prepared from Streptococcus sanguis. The synthesis of this lipid was inhibited strongly by UDP and moderately by UMP. The molar ratio of glucose to phosphate in the purified lipid was found to be 1:1. Glucose and glucose 1-phosphate were released by mild alkaline hydrolysis of the glucophospholipid. The lipid produced by mild acid degradation of the purified lipid yielded a thin-layer chromatographic profile similar to that of acid-treated undecaprenol. One of the minor components exhibited the same mobility as untreated undecaprenol. To characterize further the lipid moiety of the glucophospholipid, a polyisoprenol was purified from the neutral lipid of S. sanguis. The polyisoprenol was converted in the presence of ATP, UDP-glucose, and the particulate enzyme into a lipid which exhibited the same thin-layer chromatographic mobility as the glucophospholipid. The structure of the polyisoprenol was determined by nuclear magnetic resonance and mass spectrometry to be an undecaprenol with an internal cis-trans ratio of 7:2. These results indicate that the glucophospholipid is glucosyl monophosphoryl undecaprenol. The glucosyl moiety of the glucophospholipid was shown to be incorporated in the presence of the particulate enzyme into a macromolecule which was characterized as a lipoteichoic acid by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and DEAE-cellulose column chromatography. This result indicates that glucosyl monophosphoryl undecaprenol is the direct glucosyl donor in the synthesis of lipoteichoic acid.

scholarly journals Isolation and characterization of a γ-type phosphoinositide-specific phospholipase C (PLC-γ2)

1990 ◽  
Vol 269 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Y Homma ◽  
Y Emori ◽  
F Shibasaki ◽  
K Suzuki ◽  
T Takenawa
Keyword(s):  
Phospholipase C ◽  
Column Chromatography ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Isolation And Characterization ◽  
Delta Type ◽  
Hydrolysis Of ◽  
Cellulose Column

A novel bovine spleen phosphoinositide-specific phospholipase C (PLC) has been identified with respect to immunoreactivity with four independent antibodies against each of the PLC isoenzymes, and purified to near homogeneity by sequential column chromatography. Spleen contains three of the isoenzymes: two different gamma-types [gamma 1 and gamma 2, originally named as PLC-gamma [Rhee, Suh, Ryu & Lee (1989) Science 244, 546-550] and PLC-IV [Emori, Homma, Sorimachi, Kawasaki, Nakanishi, Suzuki & Takenawa (1989) J. Biol. Chem. 264, 21885-21890] respectively] and delta-type of the enzyme, but PLC-gamma 1 is separated from the PLC-gamma 2 pool by the first DEAE-cellulose column chromatography. Subsequently, PLC-delta is dissociated on the third heparin-Sepharose column chromatography. The purified enzyme has a molecular mass of 145 kDa on SDS/polyacrylamide-gel electrophoresis and a specific activity of 12.8 mumol/min per mg with phosphatidylinositol 4,5-bisphosphate as substrate. This enzyme activity is dependent on Ca2+ for hydrolysis of all these phosphoinositides. None of the other phospholipids examined could be its substrate at any concentration of Ca2+. The optimal pH of the enzyme is slightly acidic (pH 5.0-6.5).

scholarly journals Mouse Uterine Alkaline Phosphatase: Improved Purification by Affinity Chromatography and Further Characterization of the Enzyme

1980 ◽  
Vol 33 (3) ◽  
pp. 279 ◽  
Author(s):  
RN Murdoch ◽  
Louise E Buxton ◽  
DJ Kay
Keyword(s):  
Alkaline Phosphatase ◽  
Affinity Chromatography ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Anion Exchange Chromatography ◽  
Pregnant Mouse ◽  
Exchange Chromatography ◽  
Enzyme Preparations

An improved procedure for the purification of alkaline phosphatase from about 10 g of day 7 pregnant mouse uterine tissue is described. Following homogenization, the procedure involved solubilization and extraction with 0�8% (v/v) Triton X-lOO and 20% (v/v) n-butanol, ammonium sulfate precipitation, concanavalin A-Sepharose 4B affinity chromatography, DEAE-cellulose anion-exchange chromatography and Sephacryl S200 gel filtration. On subjecting 2162-fold purified enzyme preparations to polyacrylamide-gel electrophoresis, a single band of protein coincident with the zone of enzyme activity and having an apparent molecular weight of 205 OOO� lOOOO was identified. Affinity chromatography yielded the largest increase in purity of any step in the procedure and established the glycoprotein nature of the uterine enzyme.

PURIFICATION OF "INHIBIN" FROM HUMAN OVARIAN FOLLICULAR FLUID

1979 ◽  
Vol 90 (1) ◽  
pp. 157-166 ◽  
Author(s):  
S. Chari ◽  
C. R. N. Hopkinson ◽  
E. Daume ◽  
G. Sturm
Keyword(s):  
Follicular Fluid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Male Rats ◽  
Acetone Powder ◽  
Gel Chromatography ◽  
Apparent Homogeneity ◽  
Ovarian Follicular Fluid

ABSTRACT Following the earlier demonstration of inhibin-like activity in human ovarian follicular fluid a method for its purification to apparent homogeneity is described. The fluid was converted to acetone powder and subjected sequentially to ammonium sulphate fractionation, gel chromatography on Sephadex G-200, continuous gradient ion-exchange chromatography on DEAE-cellulose, first with a pH gradient from 8.0 to 4.0 and then with a NaCl gradient to 1 m at pH 5.2. The active fraction from this step was subjected to gel filtration on Sephadex G-100 and finally passed through an Amicon Centriflo membrane CF-25 (cut off point: 25 000 m.w.). The ultrafiltrate was homogeneous by SDS-polyacrylamide gel electrophoresis, had a molecular weight of the order of 23 000 and was capable of suppressing serum gonadotrophin levels in the castrated male rats in as low a dose as 25 μg/rat.

scholarly journals Occurrence of an endo-1,3-β-glucanase in culture fluids of the yeast Candida utilis. Purification and characterization of the enzyme activity

1979 ◽  
Vol 177 (1) ◽  
pp. 107-114 ◽  
Author(s):  
T G Villa ◽  
V Notario ◽  
J R Villanueva
Keyword(s):  
Enzyme Activity ◽  
Gel Electrophoresis ◽  
Candida Utilis ◽  
Culture Medium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Beta Glucanase ◽  
Hydrolysis Of

The endo-1,3-beta-glucanase (EC 3.2.1.6) secreted into the culture medium by cells of Candida utilis was isolated and purified to homogeneity on polyacrylamide-gel electrophoresis and in ultracentrifugation studies (s20,w = 1.97S). The purified enzyme represented only 0.001% of the total 1,3-beta-glucanase activity, the remainder being due to an exo-1,3-beta-glucanase enzyme, and behaved as an acidic glycoprotein (pI 3.3) in isoelectric-focusing experiments. The mol.wt. was estimated to be 21 000 by gel filtration and polyacrylamide-gel electrophoresis. Studies on the hydrolysis of different substrates showed that the enzyme was only able to break down (1 leads to 3)-beta-linkages, by an endo-splitting mechanism. Glucono-delta-lactone, D-glucoronolactone and heavy metal ions such as Hg2+ were inhibitors of the enzyme activity. The function of this endo-beta-glucanase in C. utilis is discussed.

Purification and characterization of the extracellular α-amylase activity of the yeast Schwanniomyces alluvius

1987 ◽  
Vol 65 (10) ◽  
pp. 899-908 ◽  
Author(s):  
F. Moranelli ◽  
M. Yaguchi ◽  
G. B. Calleja ◽  
A. Nasim
Keyword(s):  
Amino Acid ◽  
Amylase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Proteinase K ◽  
Ph Optimum ◽  
Sds Page

The extracellular α-amylase activity of the yeast Schwanniomyces alluvius has been purified by anion-exchange chromatography on DEAE-cellulose and gel-filtration chromatography on Sephadex G-100. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) and N-terminal amino acid analysis of the purified sample indicated that the enzyme preparation was homogeneous. The enzyme is a glycoprotein having a molecular mass of 52 kilodaltons (kDa) estimated by SDS–PAGE and 39 kDa by gel filtration on Sephadex G-100. Chromatofocusing shows that it is an acidic protein. It is resistant to trypsin but sensitive to proteinase K. Its activity is inhibited by the divalent cation chelators EDTA and EGTA and it is insensitive to sulfhydryl-blocking agents. Exogenous divalent cations are inhibitory as are high concentrations of monovalent salts. The enzyme has a pH optimum between 3.75 and 5.5 and displays maximum stability in the pH range of 4.0–7.0. Under the conditions tested, the activity is maximal between 45 and 50 °C and is very thermolabile. Analysis of its amino acid composition supports its acidic nature.

scholarly journals Purification and Characterization of a Novel 5-Oxoprolinase (without ATP-Hydrolyzing Activity) fromAlcaligenes faecalis N-38A

1999 ◽  
Vol 65 (2) ◽  
pp. 712-717 ◽  
Author(s):  
Atsuhisa Nishimura ◽  
Yasunori Ozaki ◽  
Hiroshi Oyama ◽  
Takashi Shin ◽  
Sawao Murao
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Alcaligenes Faecalis ◽  
Cell Extract ◽  
Homogeneous State ◽  
Amino Terminal ◽  
A Cell ◽  
Terminal Amino

ABSTRACT A novel type of 5-oxoprolinase was found in a cell extract of strain N-38A, which was later identified as Alcaligenes faecalis. The enzyme in the cell extract was purified to a homogeneous state with a yield of 16.6%. The molecular weight of the purified enzyme was estimated to be 47,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, suggesting that the enzyme is a monomeric protein. The enzyme specifically catalyzed a decyclization of l-pyroglutamate without hydrolyzing ATP and also without any requirements for metal ions such as Mg2+ and K+. The optimal pH for the decyclization was 7.4. The reaction was reversible. The equilibrium constant of the reaction, K eq = [l-glutamate]/[l-pyroglutamate], was evaluated to be approximately 0.035, which indicates that the reaction tends to form l-pyroglutamate. The amino-terminal amino acid sequence of the enzyme was H-Glu-Pro-Arg-Leu-Asp-Thr-Ser-Gln-Leu-Tyr-Ala-Asp-Val-His-Phe-. No protein with a similar sequence was found in the DNASIS database. Based on these data, it was strongly suggested that the enzyme described here is a novel type of 5-oxoprolinase.

Purification and Properties of a Neutral Protease from Soil Bacterium WM 122

1977 ◽  
Vol 37 (03) ◽  
pp. 556-565 ◽  
Author(s):  
S. E Papaioannou ◽  
W. J Marsheck
Keyword(s):  
Methyl Ester ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Soil Bacterium ◽  
Ester Hydrochloride ◽  
Apparent Homogeneity ◽  
Purification And Properties ◽  
Hydrolysis Of

SummaryAn extracellular protease SN 687, secreted by the soil bacterium isolate WM 122, has been purified by means of gel filtration, ammonium sulfate precipitation, DEAE-Sephadex and hydroxylapatite chromatography. Apparent homogeneity was ascertained by Polyacrylamide gel electrophoresis. The protease was inactivated by ethylenediamine tetracetic acid (EDTA) but not by diisopropylfluorophosphate (DFP), and it was partially inhibited by serum inhibitors. SN 687 was shown to be of high specific activity against casein and fibrin, but it did not hydrolyze L- lysine -methyl ester dihydrochloride (LME), p-tosyl-L-arginine-methyl ester hydrochloride (TAME) and N-benzoyl-L-tyrosine-ethyl ester hydrochloride (BTEE) synthetic substrates. The optimum pH for hydrolysis of casein was 7.5 and the molecular weight, as determined by gel filtration, was 31,000.

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