Purification and partial characterization of a thermostable peroxidase isoenzyme from Vigna sp seedlings

2021 ◽  
Author(s):  
Yves Mann Elate Lea Mbassi ◽  
Marie Solange Evehe Bebandoue ◽  
Wilfred Fon Mbacham ◽  
John Payne Muluh
Keyword(s):  
Thermal Inactivation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Thermal Characteristics ◽  
Heat Inactivation ◽  
Palm Tree ◽  
Great Activity ◽  
Class Iii ◽  
Peroxidase Isoenzyme ◽  
Optimum Ph

Abstract A peroxidase isoenzyme (named A6 in a previous study) was purified from radicles of a Vigna species by a combination of gel filtration on Sephadex G-100, heat treatment, CM-cellulose chromatography and DEAE-cellulose chromatography. It has been successfully separated from other anionic isoperoxidases expressed in the same tissue. It has a molecular weight of about 41 kDa and exhibits a great activity toward the oxidation of O-dianisidine, ABTS, TMB, DAB and OPD at optimum pH (pH 3 for ABTS, pH 4 for OPD and pH 6 for the others) and toward the reduction of H2O2. Its very acid optimum pH for the oxidation of ABTS is not a characteristic of other peroxidases except African oil palm tree peroxidase. Apparent Km values for these substrates were respectively 3.50 mM, 0.12 mM, 1.81 mM, 0.05 mM, 17.22 mM and 2.53 mM; catalytic efficiencies were 5.12×104 mM-1.min-1, 2.22×106 mM-1.min-1, 1.59×105 mM-1.min-1, 1.82×105 mM-1.min-1, 3.17×105 mM-1.min-1and 1.79×106 mM-1.min-1. It has an optimum temperature of activity around 60°C, and its heat inactivation fit to the first-order kinetics, with half-lives of 3.06 weeks, 13.5 hours, 15 min and 3.5 min at 50°C, 70°C, 80°C and 90°C respectively. The calculated activation energy (E) for its thermal inactivation was found to be 221.5 KJ/mol at pH8. This peroxidase isoenzyme is stable for 4 months at room temperature, loosing only 5% of its initial activity over this period. Mg2+ inhibits the activity of the enzyme. The Ca2+ ions greatly increase the stability of this peroxidase at 80°C, while Mn2+ and Zn2+ reduce it. The enzyme is inhibited by sodium azide at concentrations above 1 µM with an IC50 value around 10 µM. This inhibition, in addition to the RZ value (A403nm/A280nm) evaluated at 2.4 confirms the presence at the active site of the enzyme of a heme group common to class III peroxidases. The unusual catalytic and thermal characteristics of A6 could make it a potent tool in several biotechnological applications, especially as part of kit for enzyme immunoassays and clinical diagnosis.

Purification and characterization of exo-β-1,3-glucanase from a hatching supernatant of Strongylocentrotus intermedius

1983 ◽  
Vol 61 (1) ◽  
pp. 54-62 ◽  
Author(s):  
Kiyoshi Takeuchi
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Protein Band ◽  
Divalent Ions ◽  
Purification And Characterization ◽  
Optimum Ph ◽  
Single Protein

Exo-β-1,3-glucanase from the sea urchin embryos was purified 114-fold from the initial hatching supernatant by the following procedures: (a) gel filtration on Sephadex G-100; (b) hydrophobic chromatography on 4-phenylbutylamine-Sepharose (PBA-Sepharose); (c) two ion-exchange chromatographic steps on DEAE-cellulose; (d) gel filtration on Ultrogel AcA 34; (e) gel filtration on Sephadex G-100. The purified enzyme contained 2.2% carbohydrate and gave a single protein band corresponding to a molecular weight of 136 000 following electrophoresis on sodium dodecyl sulfate (SDS) – urea – polyacrylamide gel. Gel filtration on Ultrogel AcA 34 with a nondenaturing solvent gave a molecular weight of 130 000 ± 6000. The enzyme displayed an optimum pH at 5.0–5.5 and hydrolysed laminarin and PS(curdlan)-beads at the nonreducing ends, releasing glucose. Although activity of the purified enzyme was not affected by SDS, urea, some divalent ions, and 2-mercaptoethanol, both dithiothreitol and Hg2+ were markedly inhibitory.

scholarly journals Purification and characterization of amylase from local isolate Pseudomonas sp.SPH4

2012 ◽  
Vol 6 (1) ◽  
pp. 69-79
Author(s):  
Hala M. Ali ◽  
Ghazi M. Aziz
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Silver Ions ◽  
Enzyme Characterization ◽  
Maximum Activity ◽  
Mercury Ions ◽  
Local Isolate ◽  
Optimum Ph

The amylase produced from local isolate Pseudomonas sp. SPH4 was purified by precipitation with 30% saturation ammonium sulphate, followed by ion-exchange chromotography using DEAE-cellulose column, and Gel filtration using Sephacryl S-300 column.The two iso-enzymes (a, b) were purified to (2.83, 3.47) times in the last step with an enzymes yields of (32.36, 76.34)% respectively. Enzyme characterization of the two iso-enzymes indicated that the optimum pH for the two iso-enzymes a and b were (7, 7.5) respectively, while the optimum pH for the iso-enzymes stability were (6.5, 7) respectively. The maximum activity for iso-enzymes (a, b) appeared at 45ºC and stable for 15 min at 30-50ºC and lost approximately 50% of it's activity at rang above 75ºC. Enzyme characterization results showed that the chlorides of silver and mercury had inhibitory effect on enzyme activity, the remaining enzyme activity for the iso-enzymes (a, b) were (46.66, 36.36)% for silver ions and (41.33, 33.63)% for mercury ions at 5 mM respectively, and (28, 28.18)% for silver ions and (25.33, 19.09)% for mercury ions at 10 mM respectively. The iso-enzymes a and b were affected by chelating agent ethylene diamine tetra acetic acid (EDTA) at concentration 2mM the remaining activity (45.33, 43.63)% respectively, and 5mM the remaining activity (28, 28.18)% respectivily, and these iso-enzymes (a, b) refered to metalloenzymes. The iso-enzymes (a, b) were kept their activity when treated by reducing agent (2-mercaptoethanol) at 2 mM the remaining activity (92, 92.72)% respectively, and 5 mM the remaining activity (85.3, 89.09)% respectivily. The iso-enzymes (a, b) were kept their activity when treated by phenyl methyl sulphonyl fluoride (PMSF) at concentration 1mM the remaining activity (93.33, 90.90)% respectivily,and 5 mM the remaining activity (90.66, 87.27)% respectivily, and these indicated that these iso-enzymes didnot referred to serineamylases group.

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 Isolasi dan Karakterisasi Inulinase dari Aspergillus niger Gmn11.1 Galur Lokal

2012 ◽  
Vol 11 (1) ◽  
pp. 19 ◽  
Author(s):  
Saryono Saryono
Keyword(s):  
Molecular Weight ◽  
Aspergillus Niger ◽  
Incubation Time ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Salt Precipitation ◽  
Sds Page ◽  
Optimum Ph ◽  
Relative Molecular Weight ◽  
Optimum Ph And Temperature

Inulin is a naturally potential polysaccharide used to produced fructose and fructooligosaccharide. Inulinaseknown also as ß-fructosidase can hydrolise inulin to fructose or fructooligosaccharide. Inulinase production fromAspergillus niger Gmn11.1 isolated from dahlia tubers is conducted using medium containing 1% inulin and 0,2%yeast extract. The crude enzyme (filtrate culture) is purified by means of ammonium sulphate salt precipitation,followed by Sephadex G25 gel filtration column chromatography and DEAE cellulose anion exchanger columnchromatography. The result indicated that the enzyme had optimum pH and temperature of 4,6 and 450C, respectivelywith incubation time of 15 hours. The Km and Vmaxs values obtained from this experiment are 20 mg/ml and 0,769mg/ml/hours, respectively. Whereas the relative molecular weight of inulinase was monitored by SDS PAGE is 63KDa.

Purification and characterization of the extracellular amylases of the yeast Schwanniomyces alluvius

1984 ◽  
Vol 30 (9) ◽  
pp. 1163-1170 ◽  
Author(s):  
B. Simões-Mendes
Keyword(s):  
Deae Cellulose ◽  
Culture Fluid ◽  
Soluble Starch ◽  
Ph Stability ◽  
Starch Hydrolysis ◽  
Heat Inactivation ◽  
Optimum Temperature ◽  
Enzyme Preparations ◽  
Optimum Ph

The extracellular amylolytic system of a strain of the yeast Schwanniomyces alluvius consists of an α-amylase, a glucoamylase, and probably a debranching enzyme. Crude enzyme preparations were obtained by fractionation of the culture fluid, at the stationary phase of growth, with isopropanol. Purification was carried out by DEAE-cellulose chromatography. The glucoamylase had the following properties: molecular weight (MW), 117 000 ± 2300; optimum temperature, 50 °C; optimum pH, 4.5; range of pH stability, pH 4–6; final product of starch hydrolysis, glucose; ΔH≠ and ΔS≠ of heat inactivation, 39747 cal∙mol−1 and 274.3 cal∙deg−1∙mo−1; Km(30 °C, pH 4.5) for soluble starch, 22.22 g∙L−1. The α-amylase had the following properties: MW, 62 000 ± 500; optimum temperature, 40 °C; optimum pH, 6.3; range of pH stability, pH 4–7; final product of starch hydrolysis, maltose and glucose; ΔH≠ and ΔS≠ of heat inactivation, 36594 cal∙mol−1 and 256.9 cal∙deg−1 mol−1; Km (40 °C, pH 5.5), 2.7 g∙L−1.

Anticoagulants Produced by Thrombin from Fibrin, the Effect on Blood Coagulation, Some Physical Characteristics

1971 ◽  
Vol 26 (02) ◽  
pp. 211-223 ◽  
Author(s):  
Ch R. Muirhead ◽  
D. C Triantaphyllopoulos
Keyword(s):  
Blood Coagulation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Fibrin Clot ◽  
Disc Electrophoresis ◽  
Physical Characteristics ◽  
Advanced Stage ◽  
Kallikrein Inhibitor ◽  
Shed Blood ◽  
Complete Lysis

SummaryChromatographed thrombin in the presence of both 50 Kallikrein inhibitor units of Trasylol per ml and 0.1 M E-ACA solubilized fibrin and the products of lysis possessed anticoagulant properties. The peak of the antithrombic activity coincided with the time of complete lysis of the fibrin clot, plasmin lysed fibrin exhibited the peak of its antithrombic activity much earlier. The effect of thrombin lysed fibrin on the prothrombin consumption of shed blood was found to be inhibitory.The products of the digestion of fibrin by thrombin and by plasmin, isolated at an advanced stage of proteolysis were compared by gel filtration, disc electrophoresis and DEAE cellulose chromatography. Differences in physical characteristics of these fibrin breakdown products offer evidence that they were produced by two different enzymes.

Mechanism of Inhibitory Action on Platelet Activation of a Phospholipase A2 Isolated from Lachesis muta (Bushmaster) Snake Venom

1997 ◽  
Vol 78 (05) ◽  
pp. 1372-1380 ◽  
Author(s):  
André L Fuly ◽  
Olga L T Machado ◽  
Elias W Alves ◽  
Célia R Carlinis
Keyword(s):  
Platelet Aggregation ◽  
Enzymatic Activity ◽  
Phospholipase A2 ◽  
Inhibitory Activity ◽  
Thermal Inactivation ◽  
Anticoagulant Activity ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Crude Venom ◽  
Lachesis Muta

SummaryCrude venom from Lachesis muta exhibited procoagulant, proteolytic and phospholipase A2 activities. A phospholipase A2, denoted LM-PLA2 was purified from L. muta venom to homogeneity, through a combination of chromatographic steps involving gel-filtration on Sephacryl S-200 HR and reverse phase chromatography on a C2/C18 column. LM-PLA2 presented a single polypeptide chain with an isoelectric point at pH 4.7 and apparent molecular weight of 17 kDa. Partial aminoacid sequence indicated a high degree of homology for LM-PLA2 with other PLA2 from different sources.LM-PLA2 displayed a potent enzymatic activity as measured by indirect hemolysis of red blood cells but it was neither lethal when injected i.p. into mice nor did it present anticoagulant activity. Furthermore, LM-PLA2 displayed a moderate inhibitory activity on the aggregation of rabbit platelets induced by low levels of ADP, thrombin and arachidonate. In contrast, platelet aggregation induced by high doses of collagen was strongly inhibited by LM-PLA2 as well as ATP-release. Treatment of the protein with p-bromophenacyl bromide or 2-mercapto-ethanol, as well as thermal inactivation studies, suggested that the platelet inhibitory effect of LM-PLA2 is dependent on its enzymatic activity. Thus, the platelet inhibitory activity of LM-PLA2 was shown to be dependent on the hydrolysis of plasma phospholipids and/or lipoproteins, most probably those rich in phosphatidylcholine. Surprisingly, lyso-phosphatidylcholine released by LM-PLA2 from plasma was shown to preferentially inhibited collagen-induced platelet aggregation, in contrast to other PLA2s, whose plasma hydrolytic products indistinctly affect platelet’s response to several agonists.

INHIBITION BY HUMAN SERUM OF THE ADIPOKINETIC EFFECT OF A HUMAN PITUITARY LIPID-MOBILIZING FACTOR

1972 ◽  
Vol 71 (3) ◽  
pp. 443-453 ◽  
Author(s):  
Olav Trygstad ◽  
Irene Foss
Keyword(s):  
Human Serum ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Inhibitory Effect ◽  
Disc Electrophoresis ◽  
Inhibitory Protein ◽  
Human Pituitary ◽  
Albumin Fraction ◽  
Pituitary Glands ◽  
Normal Human

ABSTRACT A lipid-mobilizing factor (LMF) with an adipotrophic effect in human and animal fat tissue has been prepared from human pituitary glands. The addition of normal human serum to LMF reduced its lipolytic effect, and it was completely abolished by serum from a group of obese patients, whereas the lipolysis was not influenced by serum from patients with generalized lipodystrophy. By DEAE-cellulose chromatography of human serum the inhibitory effect on LMF was found to be present in a protein fraction less acidic than the main serum albumin fraction. The inhibitory fraction was deprived of some contaminants by Sephadex gel filtration. Disc electrophoresis demonstrated the presence of three components in the inhibitory protein (IP), and they were identified as albumin, transferin, and haemopexin by immuno-electrophoresis. Precipitation of these proteins by their rabbit antisera demonstrated that the inhibitory effect was present in the albumin fraction. Insulin like activity was not observed in IP. A protein binding of LMF by IP could not be demonstrated. Incubation at 37°C for one hour of a mixture of LMF and IP eliminated the electrophoretic picture of LMF. It is concluded that the inhibitory effect of human serum may be due to proteolysis of LMF.

scholarly journals Chemical identity of tryptensin with angiotensin

1980 ◽  
Vol 187 (3) ◽  
pp. 647-653 ◽  
Author(s):  
K Arakawa ◽  
M Yuki ◽  
M Ikeda
Keyword(s):  
Amino Acid ◽  
Thin Layer ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Human Plasma Protein ◽  
Plasma Protein Fraction ◽  
Layer Chromatography

Tryptensin, a vasopressor substance generated from human plasma protein fraction IV-4 by trypsin, has been isolated and the amino acid composition analysed. The procedures used for the isolation were: (a) adsorption of the formed tryptensin on Dowex 50W (X2; NH4+ form); (b) gel filtration through Sephadex G-25; (c) cation-exchange chromatography on CM-cellulose; (d) anion-exchange chromatography on DEAE-cellulose; (e) re-chromatography on CM-cellulose; (f) gel filtration on Bio-Gel P-2; (g) partition chromatography on high-pressure liquid chromatography. The homogeneity of the isolated tryptensin was confirmed by thin-layer chromatography and thin-layer electrophoresis. The amino acid analysis of the hydrolysate suggested the following proportional composition: Asp, 1; Val, 1; Ile, 1; Tyr, 1; Phe, 1; His, 1; Arg, 1; Pro, 1. This composition is identical with that of human angiotensin.

Characterization of an isozyme of S-adenosylmethionine synthetase from rat liver

1984 ◽  
Vol 62 (5) ◽  
pp. 276-279 ◽  
Author(s):  
C. H. Lin ◽  
W. Chung ◽  
K. P. Strickland ◽  
A. J. Hudson
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Enzymatic Synthesis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Aromatic Amino Acids ◽  
Adenosylmethionine Synthetase ◽  
Cellulose Column

An isozyme of S-adenosylmethionine synthetase has been purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration on a Sephadex G-200 column. The purified enzyme is very unstable and has a molecular weight of 120 000 consisting of two identical subunits. Amino acid analysis on the purified enzyme showed glycine, glutamate, and aspartate to be the most abundant and the aromatic amino acids to be the least abundant. It possesses tripolyphosphatase activity which can be stimulated five to six times by S-adenosylmethionine (20–40 μM). The findings support the conclusion that an enzyme-bound tripolyphosphate is an obligatory intermediate in the enzymatic synthesis of S-adenosylmethionine from ATP and methionine.

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