scholarly journals Characterization of the invertase from Pichia anomala

1995 ◽  
Vol 306 (1) ◽  
pp. 235-239 ◽  
Author(s):  
J Rodriguez ◽  
J A Perez ◽  
T Ruiz ◽  
L Rodriguez
Keyword(s):  
Carbon Source ◽  
Molecular Mass ◽  
Total Mass ◽  
Culture Medium ◽  
Thermal Sensitivity ◽  
Apparent Molecular Mass ◽  
Carbohydrate Moiety ◽  
Pichia Anomala

Synthesis of invertase (EC 3.2.1.26) in Pichia anomala is controlled by the carbon source in the culture medium. The enzyme was purified to homogeneity from P. anomala cells fully derepressed for invertase synthesis and shown to be a multimeric glycoprotein composed of identical subunits with an apparent molecular mass of 86.5 kDa. The carbohydrate moiety accounts for approx. 30% of the total mass of the molecule and consists of manno-oligosaccharides N-linked to the polypeptide. Most of the characteristics of the enzyme analysed in this study were similar to those previously reported for other yeast invertases, with the remarkable exception of its thermal sensitivity which appears after 15 min incubation at temperatures above 32 degrees C.

scholarly journals Purification and characterization of the invertase from Schizosaccharomyces pombe. A comparative analysis with the invertase from Saccharomyces cerevisiae

1990 ◽  
Vol 267 (3) ◽  
pp. 697-702 ◽  
Author(s):  
S Moreno ◽  
Y Sanchez ◽  
L Rodriguez
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Schizosaccharomyces Pombe ◽  
Molecular Mass ◽  
Total Mass ◽  
High Molecular Mass ◽  
Carbohydrate Moiety ◽  
Purification And Characterization ◽  
Protein Moiety ◽  
Protein Portion

Invertase (EC 3.2.1.26) was purified to homogeneity from exponentially growing cells of Schizosaccharomyces pombe fully de-repressed for synthesis of the enzyme, and was shown to be a high-molecular-mass glycoprotein that can be dissociated in the presence of 8 M-urea/1% SDS into identical subunits with an apparent molecular mass of 205 kDa. The carbohydrate moiety, accounting for 67% of the total mass, is composed of equimolar amounts of mannose and galactose. There is a small amount of glucosamine, which is probably involved in the linkage to the protein moiety, since the enzyme is sensitive to treatment with endoglycosidase H. The composition of the carbohydrate moiety resembles that found in higher-eukaryotic glycoproteins and differs from glycoproteins found in Saccharomyces cerevisiae. The protein portion of each subunit is a polypeptide of molecular mass 60 kDa, very similar to the invertase of Sacch. cerevisiae. Both proteins cross-react with antibodies raised against the protein fractions of the other, indicating that the two enzymes are similar.

scholarly journals Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

1999 ◽  
Vol 181 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Hisayo Ono ◽  
Kazuhisa Sawada ◽  
Nonpanga Khunajakr ◽  
Tao Tao ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Mass ◽  
Optimum Temperature ◽  
Sds Page ◽  
Halomonas Elongata ◽  
Optimum Ph

ABSTRACT 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had Km s of 9.1 mM forl-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.

scholarly journals Multiple carbohydrate moieties on the Na+/H+ exchanger

1993 ◽  
Vol 289 (3) ◽  
pp. 637-640 ◽  
Author(s):  
R S Haworth ◽  
O Fröhlich ◽  
L Fliegel
Keyword(s):  
Molecular Mass ◽  
Brush Border ◽  
Terminal Region ◽  
Apparent Molecular Mass ◽  
Carbohydrate Moiety ◽  
Detectable Effect ◽  
Complex Type ◽  
Brush Border Membranes ◽  
Limited Digestion ◽  
Endoglycosidase H

Affinity-purified antibodies against the C-terminal region of the Na+/H+ exchanger (NHE-1) were used to analyse the carbohydrate moiety of the protein. The Na+/H+ exchanger in human placental brush-border membranes has an apparent molecular mass of 105 kDa. Incubation of intact or detergent-solubilized membranes with glycopeptidase F removed the carbohydrate moiety and increased the apparent mobility of the exchanger. Digestion with endoglycosidase-F caused a similar change in mobility, but endoglycosidase-H had no effect, suggesting that the placental Na+/H+ exchanger is a glycoprotein of the biantennary complex type. Removal of the carbohydrate moiety with glycopeptidase F had no effect on the ability of the protein to promote the exchange of Na+ for H+, and had no detectable effect on the sensitivity of the exchanger to trypsin. Limited digestion with glycopeptidase F and neuraminidase indicated the presence of two intermediate forms between the fully glycosylated and the deglycosylated protein. This suggests the presence of at least two, and possibly three, N-linked carbohydrate moieties.

scholarly journals Expression of the glycosylphosphatidylinositol-linked complement-inhibiting protein CD59 antigen in insect cells using a baculovirus vector

1993 ◽  
Vol 295 (3) ◽  
pp. 889-896 ◽  
Author(s):  
A Davies ◽  
B P Morgan
Keyword(s):  
Molecular Mass ◽  
Culture Medium ◽  
Insect Cells ◽  
Apparent Molecular Mass ◽  
Protein Sequencing ◽  
Leader Peptide ◽  
Peptide Cleavage ◽  
Baculovirus Vector ◽  
Sds Page ◽  
Complement Lysis

CD59 antigen (CD59) is a glycosylphosphatidylinositol (GPI)-linked membrane glycoprotein which protects human cells from complement-mediated lysis. Here we report the expression of functionally active CD59 in Spodoptera frugiperda insect cells using a baculovirus vector. Recombinant CD59 was expressed abundantly on the surface of the insect cells and protected the cells from lysis by human complement. The protein was released from the cell surface by treatment with phosphatidylinositol-specific phospholipase C, indicating that it was attached to the insect cell membrane via a GPI anchor. The cells also secreted CD59 into the culture medium. Recombinant CD59 was affinity-purified from spent culture medium and from detergent extract of transfected cells. Protein purified from both sources produced multiple bands on SDS/PAGE, all of a lower apparent molecular mass than the human erythrocyte protein. However, N-terminal protein sequencing and deglycosylation studies confirmed that signals for leader peptide cleavage and N-linked glycosylation had been recognized in the insect cells, suggesting that the differences in apparent molecular mass between the native and recombinant proteins were attributable to the extent of glycosylation. Protein derived from both sources was, in part, GPI-anchored as demonstrated by phase-partition studies and incorporation into cells membranes. Incorporated recombinant protein rendered erythrocytes resistant to complement lysis.

scholarly journals Cloning, expression and characterization of YSA1H, a human adenosine 5′-diphosphosugar pyrophosphatase possessing a MutT motif

1999 ◽  
Vol 344 (2) ◽  
pp. 331-337 ◽  
Author(s):  
Lakhdar GASMI ◽  
Jared L. CARTWRIGHT ◽  
Alexander G. MCLENNAN
Keyword(s):  
Fusion Protein ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Immunoblot Analysis ◽  
Apparent Molecular Mass ◽  
Protein Glycation ◽  
Human Homologue ◽  
Protein Fragment ◽  
Sds Page

The human homologue of the Saccharomyces cerevisiae YSA1 protein, YSA1H, has been expressed as a thioredoxin fusion protein in Escherichia coli. It is an ADP-sugar pyrophosphatase with similar activities towards ADP-ribose and ADP-mannose. Its activities with ADP-glucose and diadenosine diphosphate were 56% and 20% of that with ADP-ribose respectively, whereas its activity towards other nucleoside 5′-diphosphosugars was typically 2-10%. cADP-ribose was not a substrate. The products of ADP-ribose hydrolysis were AMP and ribose 5-phosphate. Km and kcat values with ADP-ribose were 60 μM and 5.5 s-1 respectively. The optimal activity was at alkaline pH (7.4-9.0) with 2.5-5 mM Mg2+ or 100-250 μM Mn2+ ions; fluoride was inhibitory, with an IC50 of 20 μM. The YSA1H gene, which maps to 10p13-p14, is widely expressed in all human tissues examined, giving a 1.4 kb transcript. The 41.6 kDa fusion protein behaved as an 85 kDa dimer on gel filtration. After cleavage with enterokinase, the 24.4 kDa native protein fragment ran on SDS/PAGE with an apparent molecular mass of 33 kDa. Immunoblot analysis with a polyclonal antibody raised against the recombinant YSA1H revealed the presence of a protein of apparent molecular mass 33 kDa in various human cells, including erythrocytes. The sequence of YSA1H contains a MutT sequence signature motif. A major proposed function of the MutT motif proteins is to eliminate toxic nucleotide metabolites from the cell. Hence the function of YSA1H might be to remove free ADP-ribose arising from NAD+ and protein-bound poly- and mono-(ADP-ribose) turnover to prevent the occurrence of non-enzymic protein glycation.

Purification and characterization of a thermostable glucoamylase fromAspergillus fumigatus

1998 ◽  
Vol 44 (5) ◽  
pp. 493-497 ◽  
Author(s):  
William Brandani da Silva ◽  
Rosane Marina Peralta
Keyword(s):  
Aspergillus Fumigatus ◽  
Molecular Mass ◽  
Enzyme Purification ◽  
Soluble Starch ◽  
Carbohydrate Content ◽  
Apparent Molecular Mass ◽  
Starch Hydrolysis ◽  
Purification And Characterization ◽  
Original Activity

A thermostable glucoamylase from Aspergillus fumigatus was purified to homogeneity. It was a glycoprotein with 23% carbohydrate content and an apparent molecular mass of 42 kDa. The enzyme showed maximal activities at pH 4.5-5.5 and 65°C and preferentially attacked polysacharides, such as starch, glycogen, amylopectin, and amylose, rather than maltose and maltoriose. The Kmand Vmaxof soluble starch hydrolysis at 40°C and pH 5.0 were 0.1 mg ·mL-1and 161 µmol glucose equivalents liberated ·min-1·mg protein-1, respectively. The purified enzyme was remarkably insensitive to glucose. It was not affected by 500 mM D-glucose and retained about 80% of its original activity in the presence of 1000 mM of this sugar.Key words: amylase, Aspergillus fumigatus, enzyme purification, glucose insensitive, thermostableglucoamylase.

Purification and characterization of an extracellular glucoamylase from the thermophilic fungus Humicola grisea var. thermoidea

1993 ◽  
Vol 39 (9) ◽  
pp. 846-852 ◽  
Author(s):  
Luis Ricardo Orsini Tosi ◽  
Héctor Francisco Terenzi ◽  
Joāo Atílio Jorge
Keyword(s):  
Molecular Mass ◽  
Half Life ◽  
Carbohydrate Content ◽  
Thermophilic Fungus ◽  
Apparent Molecular Mass ◽  
Purification And Characterization ◽  
Sds Page ◽  
Humicola Grisea ◽  
Temperature Optima

Humicola grisea var. thermoidea mycelium grown on maltose as the main source of carbon produced at least two amylases. The major amylolytic component was purified to homogeneity and classified as a glucoamylase. The apparent molecular mass of the purified enzyme was estimated to be 63 000 Da by SDS-PAGE and 65 000 Da by Bio-Gel P-100 filtration. The purified enzyme was a glycoprotein with 1.8% carbohydrate content and pH and temperature optima of 5.0 and 55 °C, respectively. The purified glucoamylase was thermostable at 60 °C with a half-life of 16 min at 65 °C. In the presence of starch the purified enzyme retained 75% of its thermostability at 65 °C, while the addition of maltose failed to protect the activity. The purified enzyme hydrolyzed branched substrates more efficiently than linear substrates. Starch and amylopectin were the best substrates utilized and amylose was hydrolyzed faster than maltopentaose, maltotetraose, and maltotriose. Kinetic experiments suggested that maltose and starch were hydrolyzed at the same catalytic site.Key words: glucoamylase, amylase, Humicola grisea.

Morphological Characterization of Mycobacteriophage R1

1974 ◽  
Vol 32 ◽  
pp. 254-255
Author(s):  
B. L. Soloff ◽  
T. A. Rado
Keyword(s):  
Carbon Source ◽  
Stationary Phase ◽  
Growth Phase ◽  
Minimal Medium ◽  
Morphological Characterization ◽  
Logarithmic Growth Phase ◽  
Complete Lysis ◽  
Lysogenic Culture ◽  
Logarithmic Growth

Mycobacteriophage R1 was originally isolated from a lysogenic culture of M. butyricum. The virus was propagated on a leucine-requiring derivative of M. smegmatis, 607 leu−, isolated by nitrosoguanidine mutagenesis of typestrain ATCC 607. Growth was accomplished in a minimal medium containing glycerol and glucose as carbon source and enriched by the addition of 80 μg/ ml L-leucine. Bacteria in early logarithmic growth phase were infected with virus at a multiplicity of 5, and incubated with aeration for 8 hours. The partially lysed suspension was diluted 1:10 in growth medium and incubated for a further 8 hours. This permitted stationary phase cells to re-enter logarithmic growth and resulted in complete lysis of the culture.

Characterization of dissolved material during the initial phase of softwood kraft pulping

TAPPI Journal ◽  
2013 ◽  
Vol 12 (1) ◽  
pp. 37-43 ◽  
Author(s):  
HANNU PAKKANEN ◽  
TEEMU PALOHEIMO ◽  
RAIMO ALÉN
Keyword(s):  
Mass Transfer ◽  
Molecular Mass ◽  
Initial Phase ◽  
Degradation Products ◽  
Kraft Pulping ◽  
Reaction Products ◽  
Cooking Temperature ◽  
Molecular Masses ◽  
Aliphatic Carboxylic Acids

The influence of various cooking parameters, such as effective alkali, cooking temperature, and cooking time on the formation of high molecular mass lignin-derived and low molecular mass carbohydrates-derived (aliphatic carboxylic acids) degradation products, mainly during the initial phase of softwood kraft pulping was studied. In addition, the mass transfer of all of these degradation products was clarified based on their concentrations in the cooking liquor inside and outside of the chips. The results indicated that the degradation of the major hemicellulose component, galactoglucomannan, typically was dependent on temperature, and the maximum degradation amount was about 60%. In addition, about 60 min at 284°F (140°C) was needed for leveling off the concentrations of the characteristic reaction products (3,4-dideoxy-pentonic and glucoisosaccharinic acids) between these cooking liquors. Compared with low molecular mass aliphatic acids, the mass transfer of soluble lignin fragments with much higher molecular masses was clearly slower.

Incorporation of micro/nanoparticles of PCL with essential oil of Cymbopogon nardus in bacterial cellulose

2018 ◽  
Vol 1 (2) ◽  
pp. 37
Author(s):  
Aline Krindges ◽  
Vanusca Dalosto Jahno ◽  
Fernando Morisso
Keyword(s):  
Particle Size ◽  
Particulate Matter ◽  
Essential Oil ◽  
Zeta Potential ◽  
Bacterial Cellulose ◽  
Culture Medium ◽  
Static Culture ◽  
Cymbopogon Nardus ◽  
Cellulose Membranes

Incorporation studies of particles in different substrates with herbal assets growing. The objective of this work was the preparation and characterization of micro/nanoparticles containing cymbopogon nardus essential oil; and the incorporation of them on bacterial cellulose. For the development of the membranes was used the static culture medium and for the preparation of micro/nanoparticles was used the nanoprecipitation methodology. The incorporation of micro/nanoparticles was performed on samples of bacterial cellulose in wet and dry form. For the characterization of micro/nanoparticles were carried out analysis of SEM, zeta potential and particle size. For the verification of the incorporation of particulate matter in cellulose, analyses were conducted of SEM and FTIR. The results showed that it is possible the production and incorporation of micro/nanoparticles containing essential oil in bacterial cellulose membranes in wet form with ethanol.

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