scholarly journals TRANSGLYCOSYLATION ACTIVITY AND CHARACTERIZATION OF RECOMBINANT SUCROSE PHOSPHORYLASE FROM LEUCONOSTOC MESENTEROIDES MBFWRS-3(1) EXPRESSED IN ESCHERICHIA COLI

2020 ◽  
pp. 264-267
Author(s):  
EDITHA RENESTEEN ◽  
FURQON DWI CAHYO ◽  
AMARILA MALIK
Keyword(s):  
Gel Electrophoresis ◽  
Leuconostoc Mesenteroides ◽  
Sodium Dodecyl ◽  
Initial Study ◽  
Lysogeny Broth ◽  
Sucrose Phosphorylase ◽  
Transglycosylation Activity ◽  
Sds Page ◽  
Layer Chromatography

Objective: Sucrose phosphorylase (SPase) is an enzyme that catalyzes the transfer of glucosyl to various acceptor molecules. Distinct types of SPaseshave been reported, and their transglycosylase activities have been shown to differ. In general, glycosylation is a process that is used to modifybioactive compounds. As such, glycosylation can increase the chemical stability of compounds and improve their characteristics such as reduce strongsmell and sour taste. We previously cloned recombinant SPase (SPaseWRS-3[1]) from Leuconostoc mesenteroides MBFWRS-3[1] in Escherichia coli.In the current study, we aimed to characterize SPaseWRS-3 and determine its transglycosylation activity using benzoic acid (BA), ascorbic acid, andkojic acid (KA).Methods: Expression analyses were conducted in lysogeny broth (LB) medium supplemented with tetracycline and expression was induced usingisopropyl-β-d-thiogalactopyranoside. The characteristics of the 56 kDa recombinant SPase (rec-SPase) were confirmed using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Rec-SPase activity was determined spectrophotometrically using sucrose as the substrate and NADPHas the end-product at 340 nm. Transglycosylation activity was evaluated using thin-layer chromatography (TLC) on silica gel plates.Results: Our results demonstrated that the rec-SPase had an activity of 98.52% relative to the reference SPase (ref-SPase). BA and KA were determinedto undergo glucosyl transfer by rec-SPase using ref-SPase, as observed with TLC. Our findings are consistent with those reported previously for theSPase isolated from L. mesenteroides.Conclusion: Recombinant SPase activity is comparable to reference SPase activity. Our study could be the initial study to deeply observe SPase activityin other substrates as well.

scholarly journals Characterization of the Different Dextransucrase Activities Excreted in Glucose, Fructose, or Sucrose Medium byLeuconostoc mesenteroides NRRL B-1299

1998 ◽  
Vol 64 (4) ◽  
pp. 1298-1302 ◽  
Author(s):  
Marguerite Dols ◽  
M. Remaud-Simeon ◽  
R. M. Willemot ◽  
M. Vignon ◽  
P. Monsan
Keyword(s):  
Gel Electrophoresis ◽  
Leuconostoc Mesenteroides ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Traditional Culture ◽  
Culture Conditions ◽  
Acceptor Reaction ◽  
Sucrose Medium ◽  
Molecular Masses

ABSTRACT When grown in glucose or fructose medium in the absence of sucrose,Leuconostoc mesenteroides NRRL B-1299 produces two distinct extracellular dextransucrases named glucose glucosyltransferase (GGT) and fructose glucosyltransferase (FGT). The production level of GGT and FGT is 10 to 20 times lower than that of the extracellular dextransucrase sucrose glucosyltransferase (SGT) produced on sucrose medium (traditional culture conditions). GGT and FGT were concentrated by ultrafiltration before sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Their molecular masses were 183 and 186 kDa, respectively, differing from the 195 kDa of SGT. The structural analysis of the dextran produced from sucrose and of the oligosaccharides synthesized by acceptor reaction in the presence of maltose showed that GGT and FGT are two different enzymes not previously described for this strain. The polymer synthesized by GGT contains 30% α(1→2) linkages, while FGT catalyzes the synthesis of a linear dextran only composed of α(1→6) linkages.

Acid and alkaline phosphatases of Capnocytophaga species. II. Isolation, purification, and characterization of the enzymes from Capnocytophaga ochracea

1983 ◽  
Vol 29 (10) ◽  
pp. 1361-1368 ◽  
Author(s):  
Thomas P. Poirier ◽  
Stanley C. Holt
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Alkaline Phosphatases ◽  
Purification And Characterization ◽  
Molecular Weights ◽  
Sds Page ◽  
Kinetics Of

Capnocytophaga ochracea acid (AcP; EC 3.1.3.2) and alkaline (AlP; EC 3.1.3.1) phosphatase was isolated by Ribi cell disruption and purified by sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS–PAGE.) Both phosphatases eluted from Sephadex G-150 consistent with molecular weights (migration) of 140 000 and 110 000. SDS–PAGE demonstrated a 72 000 and 55 000 subunit molecular migration for AcP and AlP, respectively. The kinetics of activity of purified AcP and AIP on p-nitrophenol phosphate and phosphoseryl residues of the phosphoproteins are presented.

scholarly journals Prothrombin Tokushima: characterization of dysfunctional thrombin derived from a variant of human prothrombin

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 565-569
Author(s):  
T Inomoto ◽  
A Shirakami ◽  
S Kawauchi ◽  
T Shigekiyo ◽  
S Saito ◽  
...  
Keyword(s):  
Active Site ◽  
Binding Sites ◽  
Gel Electrophoresis ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor Xa ◽  
Molecular Defect ◽  
Sds Page

A mutant prothrombin, designated prothrombin Tokushima, was purified from plasma of a proband with 12% of normal plasma clotting activity and 42% of normal prothrombin antigen. The purified preparation gave a single band with the same mobility as that of “prothrombin” by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The factor Xa-catalyzed proteolysis of prothrombin Tokushima examined by SDS-PAGE was found to be identical to that of “prothrombin.” Subsequently thrombin Tokushima was prepared by CM-Sepharose CL-6B column chromatography after prothrombin activation by factor Xa. The molecular weight of thrombin Tokushima estimated by SDS-PAGE was identical to that of “thrombin.” Thrombin Tokushima exhibited less than 22% of normal clotting activity, and the value of kcat/Km (mumol/L-1 second-1) was less than one tenth of that of “thrombin” when Boc-Val- Pro-Arg-4-methylcoumaryl-7-amide was used as a substrate. However, active site titration using p-nitrophenyl-p′-guanidinobenzoate failed to detect any difference between the two. Thrombin Tokushima was 2.5% as effective as “thrombin” in inducing platelet aggregation. Interaction of thrombin Tokushima with antithrombin III was much slower than “thrombin” when followed by SDS-PAGE. Based on the residual thrombin activity, it was 33% as effective as “thrombin” in forming a complex with antithrombin III. These results indicate that the molecular defect resides in the thrombin portion of prothrombin Tokushima and that the binding sites for various substrates appear to be greatly impaired.

Isolation and characterization of goat serum α1-globulin protease inhibitors

1982 ◽  
Vol 60 (1) ◽  
pp. 28-35 ◽  
Author(s):  
Albert Hercz
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Protease Inhibitors ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Isolation And Characterization ◽  
Sds Page

α1-Globulin-type protease inhibitors were isolated from goat serum by two methods, namely preparative isoelectric focusing and preparative electrophoresis in polyacrylamide gel. The fractions obtained by the first method showed varying isoprotein compositions by analytical isoelectric focusing. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) revealed the presence of one protein in the fractions with the same velocity of migration as purified human α1-antitrypsin and a second protein with a slightly higher migration velocity. The ratios of trypsin-inhibiting to chymotrypsin-inhibiting capacities in all the fractions were the same and both inhibitors were stable upon storage. The reaction of the inhibitors with trypsin and chymotrypsin was also demonstrated by analytical isoelectric focusing.The fractions obtained by preparative gel electrophoresis (the second method) contained the same proteins but their proportions varied widely in different fractions as demonstrated by analytical electrofocusing in the presence of urea and by SDS–PAGE. The early fractions, which consisted predominantly of α1-antitrypsin, showed a high inhibiting capacity for trypsin and none or only negligible capacity for chymotrypsin. Conversely, in the late fractions, the proportions of the proteins and inhibiting capacities were reversed. At 4 °C the trypsin-inhibiting capacity was stable for weeks but the chymotrypsin-inhibiting capacity of the preparation rapidly decreased.These observations indicate that the inhibition of proteases by goat α1-globulins is due to at least two closely associated but distinguishable proteins. One of these, corresponding to human α1-antitrypsin, would have an appreciable capacity to inhibit trypsin, but unlike the latter, little or no capacity for chymotrypsin inhibition. The inhibition of chymotrypsin is due to the second, unidentified α1-globulin.

Characterization of coccidial proteins by two-dimensional sodium dodecyl sulphate–polyacrylamide gel electrophoresis

Parasitology ◽  
1989 ◽  
Vol 99 (2) ◽  
pp. 175-187 ◽  
Author(s):  
C. A. Sutton ◽  
M. W. Shirley ◽  
M. H. Wisher
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Two Dimensional ◽  
Sds Page

SummaryTwo dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (2D SDS–PAGE) has been used to produce ‘fingerprint’ maps of the proteins from each of the 7 species of Eimeria which infect the chicken. All 7 species could be identified from their array of polypeptides but few differences were detected between strains of the same species. Alterations to the polypeptide array associated with the stage of sporulation of the oocysts were observed. lodination of sporozoites, 2D SDS–PAGE, autoradiography and immunoblotting techniques were combined to identify polypeptides with a surface moiety and those which were antigenic.

scholarly journals Prothrombin Tokushima: characterization of dysfunctional thrombin derived from a variant of human prothrombin

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 565-569 ◽  
Author(s):  
T Inomoto ◽  
A Shirakami ◽  
S Kawauchi ◽  
T Shigekiyo ◽  
S Saito ◽  
...  
Keyword(s):  
Active Site ◽  
Binding Sites ◽  
Gel Electrophoresis ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor Xa ◽  
Molecular Defect ◽  
Sds Page

Abstract A mutant prothrombin, designated prothrombin Tokushima, was purified from plasma of a proband with 12% of normal plasma clotting activity and 42% of normal prothrombin antigen. The purified preparation gave a single band with the same mobility as that of “prothrombin” by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The factor Xa-catalyzed proteolysis of prothrombin Tokushima examined by SDS-PAGE was found to be identical to that of “prothrombin.” Subsequently thrombin Tokushima was prepared by CM-Sepharose CL-6B column chromatography after prothrombin activation by factor Xa. The molecular weight of thrombin Tokushima estimated by SDS-PAGE was identical to that of “thrombin.” Thrombin Tokushima exhibited less than 22% of normal clotting activity, and the value of kcat/Km (mumol/L-1 second-1) was less than one tenth of that of “thrombin” when Boc-Val- Pro-Arg-4-methylcoumaryl-7-amide was used as a substrate. However, active site titration using p-nitrophenyl-p′-guanidinobenzoate failed to detect any difference between the two. Thrombin Tokushima was 2.5% as effective as “thrombin” in inducing platelet aggregation. Interaction of thrombin Tokushima with antithrombin III was much slower than “thrombin” when followed by SDS-PAGE. Based on the residual thrombin activity, it was 33% as effective as “thrombin” in forming a complex with antithrombin III. These results indicate that the molecular defect resides in the thrombin portion of prothrombin Tokushima and that the binding sites for various substrates appear to be greatly impaired.

scholarly journals Characterization of plasma menbrane polypeptides of trypanosoma from bats

1989 ◽  
Vol 84 (1) ◽  
pp. 13-18 ◽  
Author(s):  
R. T. Pinho ◽  
Giovanni de Simone
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Proteins ◽  
Trypanosome Species ◽  
Cell Surface Proteins ◽  
Sds Page ◽  
Trypanosoma Dionisii ◽  
Triton X

Cell surface proteins of Trypanosoma dionisii, Trypanosoma vespertilionis and Trypanosoma sp. (M238) were radiodinated and their distribution both in the detergent-poor (DPP) and dertergent-enriched phase (DRP) was studied using a phase separation technique in Triton X-114 as well as polyacrylamide gel electrophoresis in sodium dodecyl sulphate (SDS-PAGE). Significant differences were observed in the proteins present in the DRP when the three species of trypanosoma were compared. Two major bands with 88 and 70 KDa were observed in T. sp. (M238) but were not detectable in T. dionisii and T. vespertilionis. Three polypeptides whith 96, 77 and 60 KDa were identified in the DRP of T. vespertilionis. Three major bands with 84, 72 and 60 KDa were observed in the DRP of T. dionisii. Two polypeptides with 34-36 KDa present in the DPP, were observed in the three Trypanosome species analyzed. Our observations show that T. sp. (M238) has characteristic surface polypeptides not found in T. vespertilionis.

scholarly journals Renaturation of telomere-binding proteins after the fractionation by SDS-polyacrylamide gel electrophoresis

2008 ◽  
Vol 53 (No. 7) ◽  
pp. 317-320
Author(s):  
G. Rotková
Keyword(s):  
Gel Electrophoresis ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Simple Method ◽  
Telomeric Sequences ◽  
Sds Page ◽  
Identification And Characterization

A simple method for identification and characterization of telomere-binding proteins is described in this article. After Sodium Dodecyl Sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE), proteins are eluted, renatured and used for retardation analysis with labelled oligonucleotides corresponding to human and plant of telomeric sequences. We show here that this method is efficient to recover sequence-specific DNA-binding abilities of putative telomere-binding proteins.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

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