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 Purification, characterization and radioimmunoassay of adenosine deaminase from human leukaemic granulocytes

1981 ◽  
Vol 195 (2) ◽  
pp. 389-397 ◽  
Author(s):  
D A Wiginton ◽  
M S Coleman ◽  
J J Hutton
Keyword(s):  
Molecular Weight ◽  
Adenosine Deaminase ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Periodic Acid Schiff ◽  
Apparent Homogeneity

Adenosine deaminase was purified 3038-fold to apparent homogeneity from human leukaemic granulocytes by adenosine affinity chromatography. The purified enzyme has a specific activity of 486 mumol/min per mg of protein at 35 degrees C. It exhibits a single band when subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, non-denaturing polyacrylamide-gel electrophoresis and isoelectric focusing. The pI is 4.4. The enzyme is a monomeric protein of molecular weight 44000. Both electrophoretic behaviour and molecular weight differ from those of the low-molecular-weight adenosine deaminase purified from human erythrocytes. Its amino acid composition is reported. Tests with periodic acid-Schiff reagent for associated carbohydrate are negative. Of the large group of physiological compounds tested as potential effectors, none has a significant effect. The enzyme is specific for adenosine and deoxyadenosine, with Km values of 48 microM and 34 microM respectively. There are no significant differences in enzyme function on the two substrates. erythro-9-(2-Hydroxy non-3-yl) adenine is a competitive inhibitor, with Ki 15 nM. Deoxycoformycin inhibits deamination of both adenosine and deoxyadenosine, with an apparent Ki of 60-90 pM. A specific antibody was developed against the purified enzyme, and a sensitive radioimmunoassay for adenosine deaminase protein is described.

Molecular weight determination and partial characterization of Klebsiella pneumoniae hemolysins

1986 ◽  
Vol 32 (11) ◽  
pp. 884-888 ◽  
Author(s):  
Lucila Isabel Barberis ◽  
Alberto Jorge Eraso ◽  
Maria Cristina Pàjaro ◽  
Inès Albesa
Keyword(s):  
Klebsiella Pneumoniae ◽  
Gel Electrophoresis ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Growth Media ◽  
Molecular Weight Determination ◽  
Periodic Acid Schiff

Two thiol-activated Klebsiella pneumoniae hemolysins were purified from growth media by means of salt precipitation, gel filtration, ion-exchange chromatography, and polyacrylamide gel electrophoresis. The hemolysins peaks coincided with the protein and glycoprotein peaks as determined by chromatography and electrophoresis, The molecular weights, estimated by gel filtration, were 8400 and 19 000; by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, the values were calculated as 15 500 and 27 000. The electrophoretic bands were best detected by the periodic acid–Schiff method. Reduction of the disulfide linkages did not cause the originally larger molecule to break into 8400 and 19 000 hemolysins. However, trypsin treatment cleaved the 19 000 hemolysin into an active moiety, with an electrophoretic migration similar to the 8400 hemolysin. A naturally occurring proteolytic activity was investigated using pepstatin and antipain. When the trypsin inhibitor was added to the system, the hemolytic activity was detected only in the 19 000 hemolysin and the smaller hemolysin was absent.

Cell surface characteristics of Mortierella species and their interaction with a mycoparasite

1984 ◽  
Vol 30 (3) ◽  
pp. 290-298 ◽  
Author(s):  
M. S. Manocha
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Characteristics ◽  
Protein Patterns ◽  
Periodic Acid Schiff ◽  
The Difference ◽  
Chitin And Chitosan

Cell surface characteristics of three Mortierella species differing in their response to a mycoparasite, Piptocephalis virginiana, were examined. Their cell wall composition was typical of mucoraceous fungi with chitin and chitosan as major polysaccharides. Electron microscopy revealed that the mycoparasite penetrated and formed haustoria in the hyphae of susceptible hosts, M. pusilla and M. isabellina. The failure of the parasite to establish contact and penetrate a hypha of the nonhost, M. candelabrum, was not due to cell wall thickness, rigidity, or chitin contents. Markedly different protein patterns obtained from crude alkali extracts of host and nonhost cell walls by sodium dodecyl sulfate – polyacrylamide gel electrophoresis might explain the difference in host and nonhost response to the mycoparasite. Whereas most of the bands differed only in intensity after staining with either Coomassie blue or periodic acid – Schiff reagent, there were two distinct bands of glycoproteins (76 000 and 74 000) observed in the host species which were absent in the nonhost species.

scholarly journals Platelet membrane studies in the May-Hegglin anomaly

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 279-284 ◽  
Author(s):  
BS Coller ◽  
MH Zarrabi
Keyword(s):  
Sialic Acid ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Glycoprotein ◽  
Galactose Oxidase ◽  
Periodic Acid Schiff ◽  
Giant Platelets ◽  
Total Sialic Acid ◽  
Bernard Soulier Syndrome

Abstract Since studies of the giant platelets in the Bernard-Soulier syndrome have shown decreased electrophoretic mobility, decreased sialic acid, and an abnormality in a membrane glycoprotein, we performed similar studies on the giant platelets from two patients with the May-Hegglin anomaly. The patients' platelet electrophoretic mobilities did not differ from control. Although the total sialic acid contents of the patients' platelets were greater than control when calculated per platelet, they were very similar to control when normalized for differences in platelet volume and surface area. When platelet proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis there were no differences between the glycoproteins of control and patient platelets as judged by the patterns of periodic acid Schiff staining and fluorescein-labeled concanavalin A binding. Similarly, patterns of surface glycoprotein labeling by neuraminidase/galactose oxidase/KB3H4 were identical. We conclude that unlike the giant platelets in the Bernard-Soulier syndrome, those of the May-Hegglin anomaly are not associated with a membrane abnormality detectable by these techniques.

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 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.

scholarly journals Miltenberger Class I and II erythrocytes carry a variant of glycophorin A

1983 ◽  
Vol 213 (2) ◽  
pp. 399-404 ◽  
Author(s):  
D Blanchard ◽  
A Asseraf ◽  
M J Prigent ◽  
J P Cartron
Keyword(s):  
Blood Group ◽  
Rare Variants ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Class I ◽  
Glycophorin A ◽  
M Gene ◽  
Periodic Acid Schiff ◽  
Unusual Variant

The membranes from Miltenberger Class I (Mi I) and II (Mi II) erythrocytes, two rare variants at the blood group MNSs locus, exhibited an abnormal glycoprotein of 32 kDa apparent molecular mass sharply stained by the periodic acid/Schiff procedure and a decreased content of glycoprotein alpha (synonym glycophorin A, glycoprotein MN) as seen on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Purified 125I-labelled Vicia graminea lectin binds to the unusual 32 kDa glycoprotein separated from Mi I and Mi II erythrocyte membrane of blood group NN or MN, but no significant labelling of this band was observed with Mi samples typed MM. On the basis of such lectin-labelling experiments we have described two heterozygous MN, Mi I individuals that carry one copy of an M gene producing a normal alpha-glycoprotein with M-specificity and one copy of a MiI gene producing a 32 kDa glycoprotein with N-specificity. Further investigations have shown that the 32 kDa glycoprotein was immunoprecipitated by two mouse monoclonal antibodies (R18 and R10) reacting specifically with the external domain of glycoprotein alpha. These results demonstrate that Mi I and Mi II erythrocytes carry an unusual variant of glycoprotein alpha.

scholarly journals Pj variant, a new hybrid MNSs glycoprotein of the human red-cell membrane

1982 ◽  
Vol 203 (2) ◽  
pp. 419-426 ◽  
Author(s):  
D Blanchard ◽  
J P Cartron ◽  
P Rouger ◽  
C Salmon
Keyword(s):  
Sialic Acid ◽  
Blood Group ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Low Frequency ◽  
Periodic Acid Schiff ◽  
Red Cell Membrane ◽  
Sialic Acid Content ◽  
External Domain

An unusual glycoprotein variant (Pj) was found inherited through a caucasian family exhibiting atypical N and Nvg blood-group reactivities. Pj erythrocytes are blood-group-MS homozygous and have a normal sialic acid content. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the variant contains a new component Pj of 24kDa apparent molecular mass in the monomeric state which is sharply stained by periodic acid/Schiff reagent. Both blood-group-MN (alpha) and -Ss (delta) glycoproteins were present. Homodimers (Pj2) as well as heterodimers with MN-glycoprotein (alpha Pj) and the Ss-glycoprotein (delta Pj) were also identified. The new sialoglycoprotein Pj is trypsin- and chymotrypsin-resistant in situ and carries N- and Nvg- but not M- and S-reactivities. The Pj component is labelled by lactoperoxidase-catalysed radioiodination. A 3H label is also easily introduced into the sialic acid or the galactose and galactosamine of the Pj glycoprotein. It is proposed that the Pj is a hybrid glycoprotein containing the N-terminal end of delta-glycoprotein and the C-terminal end of the alpha-glycoprotein. This proposal is supported by the finding that Pj carries a leucine residue at its N-terminus and is not immunoprecipitated by a monoclonal mouse antibody (R18) reacting specifically with the external domain of glycoprotein alpha. The red cells from the proposita Pj were found positive for a very low frequency MN antigen named Sta.

scholarly journals Identification, Purification, and Characterization of Iminodiacetate Oxidase from the EDTA-Degrading Bacterium BNC1

2001 ◽  
Vol 67 (2) ◽  
pp. 696-701 ◽  
Author(s):  
Yong Liu ◽  
Tai Man Louie ◽  
Jason Payne ◽  
Jan Bohuslavek ◽  
Harvey Bolton ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Size Exclusion ◽  
Prosthetic Group ◽  
Oxidase Gene ◽  
Nickel Affinity Chromatography ◽  
Degrading Bacterium ◽  
Apparent Homogeneity ◽  
Exclusion Chromatography

ABSTRACT Microbial degradation of synthetic chelating agents, such as EDTA and nitrilotriacetate (NTA), may help immobilizing radionuclides and heavy metals in the environment. The EDTA- and NTA-degrading bacterium BNC1 uses EDTA monooxygenase to oxidize NTA to iminodiacetate (IDA) and EDTA to ethylenediaminediacetate (EDDA). IDA- and EDDA-degrading enzymes have not been purified and characterized to date. In this report, an IDA oxidase was purified to apparent homogeneity from strain BNC1 by using a combination of eight purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band of 40 kDa, and by using size exclusion chromatography, we estimated the native enzyme to be a homodimer. Flavin adenine dinucleotide was determined as its prosthetic group. The purified enzyme oxidized IDA to glycine and glyoxylate with the consumption of O2. The temperature and pH optima for IDA oxidation were 35°C and 8, respectively. The apparent Km for IDA was 4.0 mM with a k cat of 5.3 s−1. When the N-terminal amino acid sequence was determined, it matched exactly with that encoded by a previously sequenced hypothetical oxidase gene of BNC1. The gene was expressed inEscherichia coli, and the gene product as a C-terminal fusion with a His tag was purified by a one-step nickel affinity chromatography. The purified fusion protein had essentially the same enzymatic activity and properties as the native IDA oxidase. IDA oxidase also oxidized EDDA to ethylenediamine and glyoxylate. Thus, IDA oxidase is likely the second enzyme in both NTA and EDTA degradation pathways in strain BNC1.

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.

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