scholarly journals Stability of Arthrobacterd-xylose isomerase to denaturants and heat

1992 ◽  
Vol 285 (3) ◽  
pp. 889-898 ◽  
Author(s):  
M Rangarajan ◽  
B Asboth ◽  
B S Hartley
Keyword(s):  
Gel Filtration ◽  
Xylose Isomerase ◽  
Exchange Chromatography ◽  
Reversible Dissociation ◽  
Low Concentrations ◽  
Competitive Inhibitors ◽  
Optimum Ph ◽  
Relative Stabilization ◽  
Partially Unfolded ◽  
Kinetics Of

There was no inactivation of Mg(2+)-containing Arthrobacter D-xylose isomerase up to 1 h in 0-8 M-urea at 22 degrees C, but over this range there was rapid reversible dissociation into fully active dimers with a midpoint around 4 M-urea, as shown by gradient urea gels with an activity stain, and by ion-exchange chromatography and gel filtration in urea buffers. These dimers must have the A-B* conformation, since the tetramer could dissociate into A-A*, A-B or A-B* dimer conformations, but only residues across the A-B* interface contribute to the active site. The kinetics of inactivation of the Mg(2+)-containing enzyme in 8 M-urea at higher temperatures suggest a partially unfolded Mg-A-B* dimer intermediate with 50% activity, followed by irreversible inactivation coincident with the appearance of unfolded monomer. In 0-4 M guanidinium chloride, a similar reversible dissociation into active dimers occurs, but activity falls, suggesting that A-A* and/or A-B dimers might be part of the mixture. Low concentrations of SDS also give active dimers leading to unfolded monomers, but SDS above 1% (w/v) provides relative stabilization. The apoenzyme is least thermostable (t 1/2 at 80 degrees C, pH 7, = 0.06 h) but Mg2+ stabilizes strongly (t 1/2 = 5.5 h) and Co2+ even more so. Competitive inhibitors or substrates provide a small further stabilization, but this effect is more marked at 80 degrees C, pH 5.5. Together with a marked decrease in optimum pH with temperature, this allows batch isomerizations of glucose under these conditions that produce clean but sweeter syrups.

scholarly journals The isocitrate dehydrogenases of Acinetobacter lwoffi. Separation and properties of two nicotinamide–adenine dinucleotide phosphate-linked isoenzymes

1972 ◽  
Vol 130 (1) ◽  
pp. 211-219 ◽  
Author(s):  
Colin H. Self ◽  
P. David J. Weitzman
Keyword(s):  
Molecular Weight ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Molecular Size ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Low Concentrations ◽  
Stimulation Of

Two isoenzymes of NADP-linked isocitrate dehydrogenase have been identified in Acinetobacter lwoffi and have been termed isoenzyme-I and isoenzyme-II. The isoenzymes may be separated by ion-exchange chromatography on DEAE-cellulose, by gel filtration on Sephadex G-200, or by zonal ultracentrifugation in a sucrose gradient. Low concentrations of glyoxylate or pyruvate effect considerable stimulation of the activity of isoenzyme-II. The isoenzymes also differ in pH-dependence of activity, kinetic parameters, stability to heat or urea and molecular size. Whereas isoenzyme-I resembles the NADP-linked isocitrate dehydrogenases from other organisms in having a molecular weight under 100000, isoenzyme-II is a much larger enzyme (molecular weight around 300000) resembling the NAD-linked isocitrate dehydrogenases of higher organisms.

scholarly journals Purification and Characterization of Endoglucanase from local isolate of Aspergillus flavus

2013 ◽  
Vol 10 (3) ◽  
pp. 844-853
Author(s):  
Baghdad Science Journal
Keyword(s):  
Aspergillus Flavus ◽  
Specific Activity ◽  
Gel Filtration ◽  
Temperature Stability ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Original Activity ◽  
A Value ◽  
Optimum Ph

Endoglucanase produced from Aspergillus flavus was purified by several steps including precipitation with 25 % ammonium sulphate followed by Ion –exchange chromatography, the obtained specific activity was 377.35 U/ mg protein, with a yield of 51.32 % .This step was followed by gel filtration chromatography (Sepharose -6B), when a value of specific activity was 400 U/ mg protein, with a yield of 48 %. Certain properties of this purified enzyme were investigated, the optimum pH of activity was 7 and the pH of its stability was 4.5, while the temperature stability was 40 °C for 60 min. The enzyme retained 100% of its original activity after incubation at 40 °C for 60 min; the optimum temperature for enzyme activity was 40 °C.

Studies on the ring-cyclization and ring-expansion enzymes of β-lactam biosynthesis in Cephalosporium acremonium

1983 ◽  
Vol 29 (5) ◽  
pp. 488-496 ◽  
Author(s):  
Jutta Kupka ◽  
Yong-Qiang Shen ◽  
Saul Wolfe ◽  
Arnold L. Demain
Keyword(s):  
Micrococcus Luteus ◽  
Gel Filtration ◽  
Ring Expansion ◽  
Exchange Chromatography ◽  
Cephalosporium Acremonium ◽  
Competitive Inhibitors ◽  
Isopenicillin N Synthetase ◽  
Soluble Enzymes ◽  
Temperature Optima ◽  
Isopenicillin N

Micrococcus luteus was found to be very sensitive to isopenicillin N and was used as assay organism for purification of the enzyme isopenicillin N synthetase, which cyclizes δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. Purification of the enzyme from the crude extract obtained by sonication of mycelia of Cephalosporium acremonium CW-19 was carried out by ammonium sulfate precipitation, desalting with Sephadex G-25, gel filtration on LKB ultrogel AcA44 or ion-exchange chromatography on DEAE-Sepharose. The cyclization enzyme was separated from the ring-expansion enzyme and was purified considerably more than 50-fold by this procedure. Using the purified enzyme, we found that the disulfide bis-δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine required reduction to δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine in order to behave as a substrate. The enzyme activity was stimulated by FeSO4 and ascorbate, but other cofactors, including α-ketoglutarate, were inactive. In addition to δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine, the enzyme converted adipyl-L-cysteinyl-D-valine, N-acetyl-δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine, and glycyl-δ-(L-α-aminoadipyl) L-cysteinyl-D-valine to penicillins. All of these latter peptides were competitive inhibitors of the cyclization reaction. The Km of the cyclization enzyme is 10 times higher than that of the ring-expansion enzyme, deactoxycephalosporin C synthetase. The pH and temperature optima of the two enzymes were rather similar. Phosphate inhibited ring expansion, but not cyclization. Both enzymes appear to be soluble enzymes of about 31 000 molecular weight.

Characterisation of a Neutral Protease Produced by Micrococcus luteus

1996 ◽  
Vol 51 (5-6) ◽  
pp. 429-431 ◽  
Author(s):  
M.O. Ilori ◽  
O.O. Amund ◽  
O. Omidiji
Keyword(s):  
Molecular Weight ◽  
Citric Acid ◽  
Proteolytic Enzyme ◽  
Micrococcus Luteus ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Optimum Temperature ◽  
Optimum Ph

Abstract A proteolytic enzyme produced by a cassava-ferment­ing strain of Micrococcus luteus was extracted and puri­fied 50-fold by gel filtration and ion exchange chromatography. The optimum pH for the enzyme was 7.0, the optimum temperature 25 °C, the apparent molecular weight 42 kDa and the Km value, 0.45 mg ml-1 with casein as substrate. The enzyme was stimulated by Ca2+ and Mg2+ but inhibited by Zn2+ and Co2+ ions. Other inhibitors were EDTA, KCN, citric acid and L-cysteine indicating the enzyme to be a metalloprotease.

Purification and properties of an exocellular β-glucosidase of Candida molischiana (Zikes) Meyer and Yarrow capable of hydrolyzing soluble cellodextrins

1985 ◽  
Vol 63 (11) ◽  
pp. 1160-1166 ◽  
Author(s):  
Pierre Gondé ◽  
Robert Ratomahenina ◽  
Alain Arnaud ◽  
Pierre Galzy
Keyword(s):  
Molecular Weight ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Optimum Temperature ◽  
Purification And Properties ◽  
Optimum Ph

The exocellular enzyme β-glucosidase of Candida molischiana was studied. This strain is able to ferment soluble cellodextrins. The enzyme was partially purified by ion-exchange chromatography and gel filtration. The molecular weight of this enzyme was 120 000; its optimum pH was between 4 and 4.5 and its optimum temperature was 60 °C. This enzyme was active against different soluble glucosides and was inhibited by p-chloromercuribenzoate, gluconolactone, and glucose. A "glucosyltransferase" activity appeared in the presence of ethanol. The biosynthesis of the enzyme was constitutive but repressed by glucose.

scholarly journals The cellulolytic enzymes of Botryodiplodia theobromae Pat. Separation and characterization of cellulases and β-glucosidases

1979 ◽  
Vol 177 (1) ◽  
pp. 9-19 ◽  
Author(s):  
Gabriel M. Umezurike
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Cellulolytic Enzymes ◽  
Botryodiplodia Theobromae ◽  
Molecular Weights ◽  
Low Concentrations ◽  
High Concentrations

1. Filtrates from cultures of different ages of Botryodiplodia theobromae Pat. were fractionated by gel filtration, ion-exchange chromatography and polyacrylamide-gel electrophoresis. 2. Five cellulases (C1, C2, C3, C4 and C5) were found, and their molecular weights, estimated by gel filtration, were 46000–48000 (C1), 30000–35000 (C2), 15000–18000 (C3), 10000–11000 (C4) and 4800–5500 (C5). 3. Cellulase C5 was absent from old culture filtrates. 4. Cellulase C1 had little or no activity on CM-cellulose (viscometric assay), but degraded cotton flock and Whatman cellulose powder to give cellobiose only. 5. The other components (C2–C5) produced cellobiose and smaller amounts of glucose and cellotriose from cellulosic substrates and were more active in lowering the viscosity of CM-cellulose. 6. The ratio of activities assayed by viscometry and by the release of reducing sugars from CM-cellulose increased with decrease in the molecular weights of cellulases C2–C5. 7. Cellobiose inhibited the activities of the cellulases, but glucose stimulated at low concentrations although it inhibited at high concentrations. 8. A high-molecular-weight β-glucosidase (component B1, mol.wt. 350000–380000) predominated in filtrates from young cultures, but a low-molecular-weight enzyme (B4, mol.wt. 45000–47000) predominated in older filtrates. 9. Intermediate molecular species of β-glucosidase (B2, mol.wt. 170000–180000; B3, mol.wt. 83000–87000) were also found. 10. Cellulases C2–C5 acted in synergism with C1, particularly in the presence of β-glucosidase.

scholarly journals Biosynthesis of an insulin precursor by islet tissue of cod (Gadus callarias)

1968 ◽  
Vol 110 (2) ◽  
pp. 281-288 ◽  
Author(s):  
P T Grant ◽  
K. B. M. Reid
Keyword(s):  
Gel Filtration ◽  
Molecular Size ◽  
Progressive Increase ◽  
Exchange Chromatography ◽  
Insulin Precursor ◽  
Single Polypeptide Chain ◽  
Low Concentrations ◽  
Islet Tissue ◽  
Single Polypeptide ◽  
Oxidative Sulphitolysis

1. At 15°, slices of cod islet tissue incorporated [U−14C]proline into proteins soluble in acid–ethanol at a linear rate for 6hr. 2. Initially, all the radioactivity was associated with a polypeptide that had a molecular weight of about 10000 and was appreciably more basic than cod insulin. After 1hr. there was also a significant and progressive increase in the radioactivity of insulin and of fractions intermediate in molecular size and basicity between the polypeptide and insulin. 3. O-Ethyl O-p-nitrophenyl phenylpropylphosphonate markedly decreased the radioactivity both of the intermediate fractions and of insulin, but had no significant effect on the biosynthesis of the polypeptide. In contrast, puromycin inhibited the incorporation of radioactivity into all the fractions. 4. The polypeptide had an activity of less than 0·2 international unit/mg. in the epididymal-fat-pad bioassay. Treatment with low concentrations of trypsin caused a progressive increase in the formation of an insulin-like material, judged by bioassay and ion-exchange chromatography of the digest. 5. Gel filtration of the polypeptide after oxidative sulphitolysis indicated that it was a single polypeptide chain. 6. The results suggest that the polypeptide is an insulin precursor whose formation is inhibited by puromycin and that the steps involved in the conversion of precursor into product are sensitive to O-ethyl O-p-nitrophenyl phenylpropylphosphonate.

Post-proline endopeptidase. Partial purification and characterization of the enzyme from pig kidneys

1982 ◽  
Vol 47 (4) ◽  
pp. 1139-1148 ◽  
Author(s):  
Karel Hauzer ◽  
Linda Servítová ◽  
Tomislav Barth ◽  
Karel Jošt
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Peptide Bond ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Optimum Ph ◽  
Hydrolysis Of ◽  
Prolyl Leucyl Glycinamide

Post-proline endopeptidase was isolated from pig kidneys and partially purified. The procedure consisted of fractionation with ammonium sulphate, ion exchange chromatography on DEAE-Sephadex A-50, gel filtration on Sephadex G-200 and rechromatography on DEAE-Sephadex A-50. The preparation had 55 times higher specific activity than the crude extract and did not contain any contaminating enzymic activities. The enzyme cleaved a number of proline-containing peptides and was strictly specific in catalyzing the hydrolysis of the peptide bond on the carboxyl side of the proline residue. The optimum pH for the hydrolysis of the synthetic peptides benzyl-oxycarbonylglycyl-prolyl-leucyl-glycinamide and benzyloxycarbonyl-glycyl-proline β-naphtylamide was 7.8-8.0 and, in the case of benzyloxycarbonylglycyl-proline p-nitroanilide, 7.2 to 7.5. For the hydrolysis of the tetrapeptide benzyloxycarbonylglycyl-prolyl-leucyl-glycinamide, the Km value of 75 μ mol l-1 was obtained.

scholarly journals Two acid RNases from Dactylis glomerata seeds. Purification, properties and effect of polyamines and lectins on their activity

2014 ◽  
Vol 54 (3) ◽  
pp. 241-253 ◽  
Author(s):  
Janina Wiśniowska ◽  
Bronisława Morawiecka
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Dactylis Glomerata ◽  
Hydrolytic Activity ◽  
Relative Activity ◽  
Exchange Chromatography ◽  
Lentil Lectin ◽  
Optimum Ph ◽  
Rnase Ii

Two glycoproteidic acid RNases (RNase I and RNase II) were obtained and purified from the seeds of <em>Dactylis glomerata</em> by extraction with acetate buffer, fractionation with ammonium sulfate, ion-exchange chromatography on DEAE-cellulose, DEAE-Sphadex, affinity chromatography on Con A-Sepharose and gel filtration on Bio-Gel P60. RNase I with a specific activity of 2582 U•mg<sup>-1</sup> protein and an optimum pH of 4.9 and RNase II with a specific activity of 1928 U• mg<sup>-1</sup> protein and optimum pH of 4.6, were isolated. They lacked nuclease, phosphodi- and monoesterase activities. Both forms of the enzyme hydrolyzed pyrimidine homopolymers with a preference for poly U and exhibited a low specificity for purine homopolymers (poly G and poly A). RNase I acted with a 3-fold higher hydrolytic activity on poly C homopolymer than RNase IL The hydrolytic activity of both enzymes was inhibited by Zn<sup>+2</sup>, Fe<sup>+2</sup>, Cu<sup>+2</sup> ions when yeast RNA was the substrate. The amines spermine, spermidine and tyramine at a concentration of 0.1 mM increased the enzymatic activity of both RNases by 20 to 60% of the relative activity. The hydrolytic activity of RNases I and II was stimulated by the presence of lentil lectin (LL), soybean lectin (SBA) and potato lectin (STA), and inhibited by the presence of concanavalin A. The 20-200% stimulation and 40-60% inhibition depended on the proportion, on a weight basis, of enzyme to lectin and were reversible in the presence of receptor sugars.

scholarly journals A highly inducible β-galactosidase from Enterobacter sp.

2020 ◽  
Vol 85 (5) ◽  
pp. 609-622
Author(s):  
Bestoon Shaikhan ◽  
Kemal Güven ◽  
Fatma Bekler ◽  
Ömer Acer ◽  
Reyhan Güven
Keyword(s):  
Gel Filtration ◽  
Gel Permeation Chromatography ◽  
Inhibitory Effect ◽  
Native Page ◽  
Environment And Health ◽  
Low Concentrations ◽  
Sds Page ◽  
Gel Permeation ◽  
Optimum Ph ◽  
Strong Inhibitory Effect

Enterobacter sp. 3TP2A isolated from a petroleum station was found to produce a novel, highly inducible mesophilic intracellular ?-galactosidase in the presence of lactose up to 76.5 U mg-1. The enzyme was purified to 17.3- -fold after gel permeation chromatography with a yield of approximately 11 %. The optimum pH and temperature values of the purified enzyme were found to be 8.0?9.0 and 35 ?C, respectively. The molecular weight of the enzyme was approx. 60 kDa with a single band by both SDS-PAGE and native-PAGE, and estimated by gel filtration chromatography. The enzyme was inhibited by Zn2+ and EDTA, while Cu2+ had strong inhibitory effect even at low concentrations. Activation by Mg2+ and inhibition by EDTA show that the enzyme is metal- -dependent or a metalloenzyme. The enzyme was slightly activated by 2-mercaptoethanol, while slightly inhibited by iodoacetamide. On the other hand, PCMB inhibited the enzymatic activity to a great extent, whereas it was completely inhibited by N-ethylmaleimide. The Vmax and Km values were calculated as 0.701 ?mol min-1 and 0.104 mM, respectively. The results indicated that the ?-galactosidase Enterobacter sp. 3TP2A might well be a good candidate for use in biotechnology, particularly in the area of environment and health.

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