Stabilization and purification of the secondary metabolism specific enzyme, m-hydroxybenzylalcohol dehydrogenase

1986 ◽  
Vol 32 (2) ◽  
pp. 167-175 ◽  
Author(s):  
Richard E. Scott ◽  
K. S. Lam ◽  
G. M. Gaucher
Keyword(s):  
Secondary Metabolism ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Specific Enzyme ◽  
Major Band ◽  
Exclusion Chromatography ◽  
Salt Fractionation

m-Hydroxybenzylalcohol dehydrogenase (EC 1.1.1.97), a secondary metabolism associated protein from stationary phase cultures of Penicillium urticae, was stabilized in crude extracts prior to purification. Stabilization studies resulted in the formulation of an optimal cell breakage and purification buffer. This buffer increased the enzyme's in vitro half-life at 30 °C from 14 to over 800 min which greatly aided purification and enhanced yields. Purification was achieved by salt fractionation, size-exclusion chromatography, affinity chromatography, and ion-exchange chromatography. The 1200-fold purified protein gave only one major band by sodium dodecyl sulphate – polyacrylamide gel electrophoresis.

scholarly journals Characterization of a Novel β-Galactosidase fromBifidobacterium adolescentis DSM 20083 Active towards Transgalactooligosaccharides

2000 ◽  
Vol 66 (4) ◽  
pp. 1379-1384 ◽  
Author(s):  
Katrien M. J. Van Laere ◽  
Tjakko Abee ◽  
Henk A. Schols ◽  
Gerrit Beldman ◽  
Alphons G. J. Voragen
Keyword(s):  
Size Exclusion Chromatography ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cell Extract ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Bifidobacterium Adolescentis ◽  
Exclusion Chromatography

ABSTRACT This paper reports on the effects of both reducing and nonreducing transgalactooligosaccharides (TOS) comprising 2 to 8 residues on the growth of Bifidobacterium adolescentis DSM 20083 and on the production of a novel β-galactosidase (β-Gal II). In cells grown on TOS, in addition to the lactose-degrading β-Gal (β-Gal I), another β-Gal (β-Gal II) was detected and it showed activity towards TOS but not towards lactose. β-Gal II activity was at least 20-fold higher when cells were grown on TOS than when cells were grown on galactose, glucose, and lactose. Subsequently, the enzyme was purified from the cell extract of TOS-grown B. adolescentis by anion-exchange chromatography, adsorption chromatography, and size-exclusion chromatography. β-Gal II has apparent molecular masses of 350 and 89 kDa as judged by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating that the enzyme is active in vivo as a tetramer. β-Gal II had an optimal activity at pH 6 and was not active below pH 5. Its optimum temperature was 35°C. The enzyme showed highestV max values towards galactooligosaccharides with a low degree of polymerization. This result is in agreement with the observation that during fermentation of TOS, the di- and trisaccharides were fermented first. β-Gal II was active towards β-galactosyl residues that were 1→4, 1→6, 1→3, and 1↔1 linked, signifying its role in the metabolism of galactooligosaccharides by B. adolescentis.

scholarly journals Purification and properties of heparin-releasable lipoprotein- associated coagulation inhibitor

Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 394-400 ◽  
Author(s):  
WF Novotny ◽  
M Palmier ◽  
TC Wun ◽  
GJ Jr Broze ◽  
JP Miletich
Keyword(s):  
Vascular Endothelium ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Major Band ◽  
Coagulation Inhibitor

The lipoprotein-associated coagulation inhibitor (LACI) is present in vivo in at least three different pools: sequestered in platelets, associated with plasma lipoproteins, and released into plasma by intravenous heparin, possibly from vascular endothelium. In this study we have purified the heparin-relesable form of LACI from post-heparin plasma and show that it is structurally different from lipoprotein LACI. The purification scheme uses heparin-agarose chromatography, immunoaffinity chromatography, and size-exclusion chromatography and results in a 185,000-fold purification with a 33% yield. Heparin- releasable LACI (HRL), as analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, under reducing conditions, appears as a major band at 40 Kd and a minor band at 36 Kd. Immunoblot analysis suggests that the 36-Kd band arises from carboxyl-terminus proteolysis that occurs during the purification. HRL has a specific activity similar to that of HepG2 or lipoprotein LACI. HRL and lipoprotein LACI combine with lipoproteins in vitro while purified HepG2 LACI does not. I125-labeled HRL, injected into a rabbit, is cleared more slowly than I125-labeled HepG2 LACI, which may be due to attachment to lipoproteins in vivo. Preliminary evidence suggests that HRL is associated with vascular endothelium, possibly by attachment to glycosaminoglycans.

scholarly journals A purified lectin with larvicidal activity from a woodland mushroom, Agaricus semotus Fr.

2021 ◽  
Vol 65 (1) ◽  
pp. 65-73
Author(s):  
Isaiah O. Adedoyin ◽  
Taiwo S. Adewole ◽  
Titilayo O. Agunbiade ◽  
Francis B. Adewoyin ◽  
Adenike Kuku
Keyword(s):  
Larvicidal Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Complete Inactivation ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Effects Of Temperature

This study investigated the larvicidal activity on Culex quinquefasciatus of lectin purified from fresh fruiting bodies of woodland mushroom, Agaricus semotus. A. semotus lectin (ASL) was purified via ion-exchange chromatography on DEAE-cellulose A-25 and size exclusion chromatography on Sephadex G-100 matrix. Molecular weight (16.6 kDa) was estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The effects of temperature, pH, metal chelation- and larvicidal activity of ASL were also investigated. The ASL indifferently agglutinated the erythrocytes of the human ABO blood system and was stable at acidic pH and below 50 °C whereas 66% of its activity was lost at 60 °C with complete inactivation at 70 °C. ASL is a metalloprotein requiring barium ion as chelation of metals by 50 mM EDTA rendered the lectin inactive, while the addition of BaCl2, among other metal salts, restored the activity. ASL showed larvicidal activity against C. quinquefasciatus larvae after 24 h with a mortality of 5 and 95% at 5 and 25 mg/mL respectively, and LC50 of 13.80 mg/mL. This study concluded that purified A. semotus lectin showed impressive larvicidal activity, which could be exploited in its development as an insecticidal agent.

scholarly journals Purification and properties of heparin-releasable lipoprotein- associated coagulation inhibitor

Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 394-400 ◽  
Author(s):  
WF Novotny ◽  
M Palmier ◽  
TC Wun ◽  
GJ Jr Broze ◽  
JP Miletich
Keyword(s):  
Vascular Endothelium ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Major Band ◽  
Coagulation Inhibitor

Abstract The lipoprotein-associated coagulation inhibitor (LACI) is present in vivo in at least three different pools: sequestered in platelets, associated with plasma lipoproteins, and released into plasma by intravenous heparin, possibly from vascular endothelium. In this study we have purified the heparin-relesable form of LACI from post-heparin plasma and show that it is structurally different from lipoprotein LACI. The purification scheme uses heparin-agarose chromatography, immunoaffinity chromatography, and size-exclusion chromatography and results in a 185,000-fold purification with a 33% yield. Heparin- releasable LACI (HRL), as analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, under reducing conditions, appears as a major band at 40 Kd and a minor band at 36 Kd. Immunoblot analysis suggests that the 36-Kd band arises from carboxyl-terminus proteolysis that occurs during the purification. HRL has a specific activity similar to that of HepG2 or lipoprotein LACI. HRL and lipoprotein LACI combine with lipoproteins in vitro while purified HepG2 LACI does not. I125-labeled HRL, injected into a rabbit, is cleared more slowly than I125-labeled HepG2 LACI, which may be due to attachment to lipoproteins in vivo. Preliminary evidence suggests that HRL is associated with vascular endothelium, possibly by attachment to glycosaminoglycans.

scholarly journals Prebiotic Activity of Poly- and Oligosaccharides Obtained from Plantago major L. Leaves

2020 ◽  
Vol 10 (8) ◽  
pp. 2648 ◽  
Author(s):  
Paolina Lukova ◽  
Mariana Nikolova ◽  
Emmanuel Petit ◽  
Redouan Elboutachfaiti ◽  
Tonka Vasileva ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Galacturonic Acid ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Plantago Major ◽  
Prebiotic Activity ◽  
Molecular Fraction ◽  
Exclusion Chromatography

The aim of the present study was to evaluate the prebiotic potential of Plantago major L. leaves water-extractable polysaccharide (PWPs) and its lower molecular fractions. The structure of PWPs was investigated by high pressure anion exchange chromatography (HPAEC), size exclusion chromatography coupled with multi-angle laser light scattering detector (SEC-MALLS) and Fourier-transform infrared (FTIR) spectroscopy. The chemical composition and monosaccharide analyses showed that galacturonic acid was the main monosaccharide of PWPs followed by glucose, arabinose, galactose, rhamnose and xylose. FTIR study indicated a strong characteristic absorption peak at 1550 cm−1 corresponding to the vibration of COO− group of galacturonic acid. The PWPs was subjected to hydrolysis using commercial enzymes to obtain P. major low molecular fraction (PLM) which was successively separated by size exclusion chromatography on Biogel P2. PWPs and PLM were examined for in vitro prebiotic activity using various assays. Results gave evidence for changes in optical density of the bacteria cells and pH of the growth medium. A heterofermentative process with a lactate/acetate ratio ranged from 1:1 to 1:5 was observed. The ability of PLM to stimulate the production of certain probiotic bacteria glycohydrolases and to be fermented by Lactobacillus sp. strains was successfully proved.

scholarly journals Impact of Botrytis cinerea Contamination on the Characteristics and Foamability of Yeast Macromolecules Released during the Alcoholic Fermentation of a Model Grape Juice

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 472
Author(s):  
Richard Marchal ◽  
Thomas Salmon ◽  
Ramon Gonzalez ◽  
Belinda Kemp ◽  
Céline Vrigneau ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Alcoholic Fermentation ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Grape Juice ◽  
Size Exclusion ◽  
Periodic Acid Schiff ◽  
Exclusion Chromatography ◽  
The Impact

Botrytis cinerea is a fungal pathogen responsible for the decrease in foamability of sparkling wines. The proteolysis of must proteins originating from botrytized grapes is well known, but far less information is available concerning the effect of grape juice contamination by Botrytis. The impact from Botrytis on the biochemical and physico-chemical characteristics of proteins released from Saccharomyces during alcoholic fermentation remains elusive. To address this lack of knowledge, a model grape juice was inoculated with three enological yeasts with or without the Botrytis culture supernatant. Size exclusion chromatography coupled to multi-angle light scattering (SEC-MALLS) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) techniques (AgNO3 and periodic acid Schiff staining) was used in the study. When Botrytis enzymes were present, a significant degradation of the higher and medium MW molecules released by Saccharomyces was observed during alcoholic fermentation whilst the lower MW fraction increased. For the three yeast strains studied, the results clearly showed a strong decrease in the wine foamability when synthetic musts were inoculated with 5% (v/v) of Botrytis culture due to fungus proteases.

scholarly journals The 47-kD fragment of talin is a substrate for protein kinase P

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3343-3349 ◽  
Author(s):  
PC Simons ◽  
L Elias
Keyword(s):  
Protein Kinase ◽  
Ion Exchange ◽  
Sequence Data ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Myelogenous Leukemia ◽  
Blue Staining ◽  
Major Protein

Abstract This laboratory has been characterizing protein serine/threonine kinase reactions of hematopoietic tissues, whose most distinguishing characteristics in vitro are stimulation with vesicular phosphatidyl glycerol, and the ability to function using Mn2+ as the sole divalent cation. The major protein substrates are a 73-kD protein and a protein migrating near ovalbumin on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 47-kD protein was partially purified from cells harvested by leukapheresis from a patient with acute myelogenous leukemia, using ammonium sulfate precipitation and ion exchange chromatography. This partially purified ion-exchange fraction contained an endogenous kinase activity with characteristics similar to those we previously described of protein kinase P (protein kinase, phospholipid- stimulable: PK-P), but not typical of any form of protein kinase C (PK- C). With longer phosphorylation, the 47-kD band showed increasingly lower mobility demonstrable both by Coomassie blue staining and autoradiography, suggesting both that it was multiply phosphorylated, and that the excisable band was pure. The protein was thus eluted from preparative gel slices and digested with endoproteinase lys C. Sequence data from the fragments identified the protein as the 47-kD calpain fragment of talin, a protein found in focal adhesion plaques and some cell-cell contacts. PK-C phosphorylated the 47-kD protein, as has been reported previously, and phosphopeptide mapping disclosed a similar pattern of phosphorylation using either PK-C or the endogenous activity. The 47-kD protein labeled with the endogenous kinase contained predominantly phosphoserine, with some phosphothreonine and a trace of phosphotyrosine. Intact, purified talin was also phosphorylated by PK-P in a phospholipid-stimulable manner, but at 1/20 the rate of the 47-kD fragment.

Effects of Some Vitamins on Lactoperoxidase Enzyme Activity

2009 ◽  
Vol 79 (3) ◽  
pp. 188-194 ◽  
Author(s):  
Melda Sisecioglu ◽  
Murat Cankaya ◽  
Hasan Ozdemir
Keyword(s):  
Ascorbic Acid ◽  
Enzyme Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bovine Milk ◽  
Exchange Chromatography ◽  
Inhibition Mechanism ◽  
Vitamin K3 ◽  
Vitro Effect

Objective: The present paper investigates the in vitro effect of L-ascorbic acid (vitamin C), menadione sodium bisulfate (vitamin K3), and folic acid on purified lactoperoxidase (LPO). Methods: This enzyme was purified from bovine milk by Amberlite CG 50 resin, CM Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography. Results: Rz (A412/A280) value for the purified LPO was found to be 0.8. Lactoperoxidase was purified 20.45-fold with a yield of 28.8 %. Purity of enzyme was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method and a single band was observed. All tested vitamins caused inhibition of the enzyme activity and displayed a competitive type of inhibition mechanism. IC50 values of these three vitamins were 2.03 µM, 0.025 mM, and 0.0925 mM, and the Ki constants were 0.508±0.257 µM, 0.0107±0.0044 mM, and 0.0218±0.0019 mM respectively. Conclusion: The vitamins discussed here displayed inhibition-type competition with LPO enzyme at varying concentrations. Our study showed that L-ascorbic acid exhibited a much higher inhibitory effect at lower concentrations, so it was evidently a more potent inhibitor than other vitamins tested.

scholarly journals Purification and partial biochemical characterization of normal human interleukin 1.

1984 ◽  
Vol 160 (3) ◽  
pp. 772-787 ◽  
Author(s):  
J A Schmidt
Keyword(s):  
High Performance ◽  
Biochemical Characterization ◽  
Interleukin 1 ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Thymocyte Proliferation ◽  
Exclusion Chromatography ◽  
Normal Human

A protocol for the rapid, efficient purification of the major charged species of human interleukin 1 (IL-1) has been developed using high performance anion exchange and size exclusion chromatography. The isolated material is pure as determined by sodium dodecyl sulfate (SDS) gradient polyacrylamide gel electrophoresis (PAGE) and analytical isoelectric focusing (IEF). The molecular weight of the purified material is 15,000 and the isoelectric point (pI) is 6.8, values that are in good agreement with those previously reported for human IL-1. 10(-10) M concentrations of the purified material give half-maximal stimulation in the thymocyte proliferation assay. Amounts of IL-1 sufficient for receptor studies and detailed biochemical analysis can now be produced on a regular basis.

Purification and partial characterization of a rat kidney aldehyde dehydrogenase that oxidizes retinal to retinoic acid

1993 ◽  
Vol 71 (1-2) ◽  
pp. 85-89 ◽  
Author(s):  
Jean Labrecque ◽  
Pangala V. Bhat ◽  
André Lacroix
Keyword(s):  
Molecular Weight ◽  
Retinoic Acid ◽  
Vitamin A ◽  
Aldehyde Dehydrogenase ◽  
Rat Kidney ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Exchange Chromatography ◽  
Size Exclusion

A NAD-dependent aldehyde dehydrogenase (EC 1.2.1.3) which catalyzes the oxidation of retinal to retinoic acid was purified to homogeneity from rat kidney by using Affi-Gel blue affinity chromatography and chromatofocusing, followed by Mono-Q anion-exchange chromatography. The apparent molecular weight of the native enzyme determined by size-exclusion fast protein liquid chromatography was 140 000. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis gave a subunit molecular weight of 53 000. The isoelectric point as measured by chromatofocusing was 8.5. The enzyme also catalyzed the oxidation of acetaldehyde, but showed much lower Km value for the retinal substrate. We suggest that aldehyde dehydrogenase found in the kidney may be a specific retinal dehydrogenase, involved in vitamin A metabolism.Key words: aldehyde dehydrogenase, vitamin A, retinal, retinoic acid, kidney.

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