Cellobiohydrolase from Trichoderma reesei

A 1,4-beta-D-glucan cellobiohydrolase (EC 3.2.1.91) was purified from the culture liquid of Trichoderma reesei by using biospecific sorption on amorphous cellulose and immunoaffinity chromatography. A single protein band in polyacrylamide-gel electrophoresis and one arc in immunoelectrophoresis corresponded to the enzyme activity. The Mr was 65 000. The pI was 4.2-3.6. The purified enzyme contained about 10% hexose. The enzyme differs from previously described cellobiohydrolases in being more effective in the hydrolysis of cellulose.

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Glycosidases induced in Aspergillus tamarii. Secreted α-d-galactosidase and β-d-mannanase

Biochemical Journal ◽

10.1042/bj2190857 ◽

1984 ◽

Vol 219 (3) ◽

pp. 857-863 ◽

Cited By ~ 53

Author(s):

A Civas ◽

R Eberhard ◽

P Le Dizet ◽

F Petek

Keyword(s):

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Deae Cellulose ◽

Preceding Paper ◽

Protein Band ◽

Polyacrylamide Gel ◽

Aspergillus Tamarii ◽

Electrophoretic Method ◽

Single Protein

An alpha-D-galactosidase (EC 3.2.1.22) and a beta-D-mannanase (EC 3.2.1.78), which were secreted into the growth medium when Aspergillus tamarii was cultivated in the presence of galactomannan, were purified by a procedure including chromatography on hydroxyapatite and DEAE-cellulose columns. Each of these enzymes showed a single protein band, corresponding to their respective activities, on polyacrylamide-gel electrophoresis. Both enzymes were shown to be glycoproteins containing N-acetylglucosamine, mannose and galactose, with molar proportions of 1:6:1.5 for alpha-D-galactosidase and 1:13:8 for beta-D-mannanase. Mr values as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and by the electrophoretic method of Hedrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164] were 56000 and 53000 respectively. The alpha-D-galactosidase differed markedly from the mycelial forms I and II studied in the preceding paper [Civas, Eberhard, Le Dizet & Petek (1984) Biochem. J. 219, 849-855] with regard to both its kinetic and structural properties.

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Phosphorylation of partially purified 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase from rat spleen

Biochemical Journal ◽

10.1042/bj2450893 ◽

1987 ◽

Vol 245 (3) ◽

pp. 893-897 ◽

Cited By ~ 23

Author(s):

J Gomez-Cambronero ◽

J M Mato ◽

F Vivanco ◽

M Sanchez-Crespo

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Protein Band ◽

Improved Method ◽

Serine Residue ◽

Dependent Protein Kinase ◽

Single Protein ◽

Dependent Protein

A new improved method for purification of the enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase (EC 2.3.1.67) from rat spleen is described. The catalytic subunit of cyclic AMP-dependent protein kinase in the presence of MgATP stimulated about 3-fold the activity of this partially purified enzyme activity. When [gamma-32P]ATP was included in the assay mixture, the analysis of phosphoprotein products by SDS/polyacrylamide-gel electrophoresis and autoradiography showed the incorporation of [32P]phosphate into a single protein band of about 30 kDa. Analysis of the phosphorylated amino acids indicated that the phosphate was incorporated into a serine residue. Activation of the acetylation reaction by the protein kinase was reversible. The reversal of the activation was coincident with the loss of the [32P]phosphate incorporated into the 30 kDa protein band, which suggests that the acetyltransferase is regulated by a phosphorylation-dephosphorylation mechanism dependent on cyclic AMP.

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Glycosidases induced in Aspergillus tamarii. Mycelial α-d-galactosidases

Biochemical Journal ◽

10.1042/bj2190849 ◽

1984 ◽

Vol 219 (3) ◽

pp. 849-855 ◽

Cited By ~ 23

Author(s):

A Civas ◽

R Eberhard ◽

P Le Dizet ◽

F Petek

Keyword(s):

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Deae Cellulose ◽

Protein Band ◽

Polyacrylamide Gel ◽

Aspergillus Tamarii ◽

Single Protein Band ◽

Single Protein ◽

Alpha Galactosidase

Two alpha-D-galactosidases (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) produced by Aspergillus tamarii were purified from the mycelial extract by a procedure including chromatography on hydroxyapatite, DEAE-cellulose and ECTEOLA-cellulose. Each of these enzymes showed a single protein band corresponding to the alpha-D-galactosidase activity when examined by polyacrylamide-gel electrophoresis. They catalysed the hydrolysis of o-nitrophenyl alpha-D-galactoside, melibiose, raffinose and stachyose, but did not attack the galactomannans. Their Mr values were respectively 265000 +/- 5000 and 254000 +/- 5000 by the method of Hedrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164]. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate in each case showed a single protein band, with Mr 88000 and 77500 respectively. The purified enzymes contained carbohydrate, consisting of N-acetylglucosamine, mannose, glucose and galactose in the estimated molar proportions of 1:9:5:8 in alpha-galactosidase I.

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Conditions of hydrolysis with a specific pair of endo- and exo-cellulases

10.32920/ryerson.14652435.v1 ◽

2021 ◽

Author(s):

Yoshiki Kitano

Keyword(s):

Synergistic Effect ◽

Crystalline Cellulose ◽

Carbohydrate Binding ◽

Amorphous Cellulose ◽

Potential Synergy ◽

Hydrolysis Of Cellulose ◽

Potential Alternative ◽

Specific Pair ◽

Hydrolysis Of ◽

Increased Activity

Enzymatic hydrolysis of cellulose is a technology involved in the production of bioethanol, a potential alternative renewable energy. Many cellulases with endo- and exo- type of activity are known to hydrolyze cellulose synergistically. In this thesis, potential synergy between an endo-cellulase, Cel5B, with and without a carbohydrate- binding module (CBM6), and a new exo-cellulase, CBH1, from Trichoderma harzianum FP108 were examined during the hydrolysis of semi- crystalline cellulose (Avicel). Since CBM6 is recognized as having a high affinity for amorphous cellulose, it was hypothesized that this affinity could enhance the synergistic effect between the endo- and exo-cellulases by focusing the action to Cel5B+CBM6 on the amorphous regions of the Avicel substrate. The increased activity of Cel5B+CBM6 over Cel5B alone was confirmed. However, in contrast to our expectations, a synergistic effect was not observed between either endo- and exo-cellulase pairs. From the obtained hydrolysis yield, it was inferred that Cel5B+CBM6 may have exo-type activity that caused a competitive interaction with the exo-cellulase, which resulted in no synergy.

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Production of Recombinant Trichoderma reesei Cellobiohydrolase II in a New Expression System Based on Wickerhamomyces anomalus

Enzyme Research ◽

10.1155/2017/6980565 ◽

2017 ◽

Vol 2017 ◽

pp. 1-8 ◽

Cited By ~ 3

Author(s):

Dennis J. Díaz-Rincón ◽

Ivonne Duque ◽

Erika Osorio ◽

Alexander Rodríguez-López ◽

Angela Espejo-Mojica ◽

...

Keyword(s):

Trichoderma Reesei ◽

Recombinant Expression ◽

Expression System ◽

Enzymatic Extract ◽

Native Strain ◽

Wickerhamomyces Anomalus ◽

Optimum Ph ◽

Hydrolysis Of Cellulose ◽

Cellobiohydrolase Ii ◽

Hydrolysis Of

Cellulase is a family of at least three groups of enzymes that participate in the sequential hydrolysis of cellulose. Recombinant expression of cellulases might allow reducing their production times and increasing the low proteins concentrations obtained with filamentous fungi. In this study, we describe the production of Trichoderma reesei cellobiohydrolase II (CBHII) in a native strain of Wickerhamomyces anomalus. Recombinant CBHII was expressed in W. anomalus 54-A reaching enzyme activity values of up to 14.5 U L−1. The enzyme extract showed optimum pH and temperature of 5.0–6.0 and 40°C, respectively. Enzyme kinetic parameters (KM of 2.73 mM and Vmax of 23.1 µM min−1) were between the ranges of values reported for other CBHII enzymes. Finally, the results showed that an enzymatic extract of W. anomalus 54-A carrying the recombinant T. reesei CBHII allows production of reducing sugars similar to that of a crude extract from cellulolytic fungi. These results show the first report on the use of W. anomalus as a host to produce recombinant proteins. In addition, recombinant T. reesei CBHII enzyme could potentially be used in the degradation of lignocellulosic residues to produce bioethanol, based on its pH and temperature activity profile.

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Isolation and Composition of the Seed Globulins of Winged Bean, Psophocarpus tetragonolobus (L.) DC

Functional Plant Biology ◽

10.1071/pp9780357 ◽

1978 ◽

Vol 5 (3) ◽

pp. 357 ◽

Cited By ~ 11

Author(s):

JM Gillespie ◽

RJ Blagrove

Keyword(s):

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Winged Bean ◽

The Other ◽

Bean Seed ◽

Psophocarpus Tetragonolobus ◽

Disulfide Bonding ◽

Single Protein ◽

Seed Globulins ◽

Sulfur Containing

The amino acid composition of winged bean seed meal is similar to that of soybean but their storage globulins are quite different. Winged bean proteins are soluble to the extent of 60% at the pH of a meal-water slurry (pH 6.6), 80% at pH 11 but only 12% at pH 5. However, the proteins are soluble to the extent of 80% from pH 5 to 9 in 10% NaCl rising to 90% at pH 11. There are no satisfactory ways of recovering all the proteins from solution by simple changes in pH or ionic strength. Winged bean seed contains major proteins with sedimentation coefficients of 2 S and 6 S. Electrophoresis on cellulose acetate resolves three globulin fractions which we have named psophocarpins A, B, and C. The proteins from these electrophoretic regions have been isolated and partially purified. Psophocarpin A is essentially a single protein comparatively rich in sulfur-containing amino acids while the other fractions are composed of a number of related components which have not been separated. When examined by SDS-polyacrylamide gel electrophoresis, the globulin fractions differed in the kind of subunit proteins they contain and in the extent of disulfide bonding. The 40 000 mol. wt subunit of psophocarpin A contains disulfide bonded chains of mol. wt 16 000 and 24 000. The proteins corresponding to the other electrophoretic regions are more complex.

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The formation of lipid-linked sugars by cell-free preparations of lactating rabbit mammary gland

Biochemical Journal ◽

10.1042/bj1700479 ◽

1978 ◽

Vol 170 (3) ◽

pp. 479-486 ◽

Cited By ~ 4

Author(s):

D A White

Keyword(s):

Mammary Gland ◽

Acid Hydrolysis ◽

Alkaline Hydrolysis ◽

Gel Electrophoresis ◽

Incubation Medium ◽

Polyacrylamide Gel Electrophoresis ◽

Protein Fractions ◽

Exogenous Substrate ◽

Major Species ◽

Hydrolysis Of

1. A lactating rabbit mammary-gland microsomal system catalysed the incorporation of mannose from GDP-[U-14C]mannose into three endogenous acceptors, (i) polyprenyl phosphate mannose, (ii) lipid-linked oligosaccharide and (iii) protein. 2. Synthesis of polyprenyl phosphate mannose was stimulated by addition of dolichol phosphate to the incubation medium and was reversed by addition of GDP. The product had properties identical with those of authentic dolichol phosphate mannose. 3. The oligosaccharides derived from acid hydrolysis of the lipid-linked oligosaccharide fraction were of six, eight and nine to ten monosaccharide units, the octasaccharide being the major species formed. The oligosaccharide appeared to be attached to the lipid via a pyrophosphate bridge, since strong alkaline hydrolysis liberated an oligosaccharide phosphate. 4. Polyprenyl phosphate mannose served as a mannose donor to lipid-linked oligosaccharides and protein. When added as exogenous substrate it gave rise to a lipid-linked oligosaccharide of about six units. 5. Incorporation of radioactivity in protein was low, but polyacrylamide-gel electrophoresis of the protein fractions indicated that polypeptides of mol.wts. 115000, 75000 and 33000 were labelled.

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Occurrence of an endo-1,3-β-glucanase in culture fluids of the yeast Candida utilis. Purification and characterization of the enzyme activity

Biochemical Journal ◽

10.1042/bj1770107 ◽

1979 ◽

Vol 177 (1) ◽

pp. 107-114 ◽

Cited By ~ 16

Author(s):

T G Villa ◽

V Notario ◽

J R Villanueva

Keyword(s):

Enzyme Activity ◽

Gel Electrophoresis ◽

Candida Utilis ◽

Culture Medium ◽

Polyacrylamide Gel Electrophoresis ◽

Gel Filtration ◽

Polyacrylamide Gel ◽

Beta Glucanase ◽

Hydrolysis Of

The endo-1,3-beta-glucanase (EC 3.2.1.6) secreted into the culture medium by cells of Candida utilis was isolated and purified to homogeneity on polyacrylamide-gel electrophoresis and in ultracentrifugation studies (s20,w = 1.97S). The purified enzyme represented only 0.001% of the total 1,3-beta-glucanase activity, the remainder being due to an exo-1,3-beta-glucanase enzyme, and behaved as an acidic glycoprotein (pI 3.3) in isoelectric-focusing experiments. The mol.wt. was estimated to be 21 000 by gel filtration and polyacrylamide-gel electrophoresis. Studies on the hydrolysis of different substrates showed that the enzyme was only able to break down (1 leads to 3)-beta-linkages, by an endo-splitting mechanism. Glucono-delta-lactone, D-glucoronolactone and heavy metal ions such as Hg2+ were inhibitors of the enzyme activity. The function of this endo-beta-glucanase in C. utilis is discussed.

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The Nα-acetylenkephalin carboxypeptidase activity of N-acetyltyrosine deacetylase from monkey kidney. Purification, characterization and substrate specificity

Biochemical Journal ◽

10.1042/bj2090471 ◽

1983 ◽

Vol 209 (2) ◽

pp. 471-479 ◽

Cited By ~ 3

Author(s):

S T George ◽

A S Balasubramanian

Keyword(s):

Amino Acids ◽

Gel Electrophoresis ◽

High Speed ◽

Metal Chelate ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Protein Band ◽

Monkey Kidney ◽

Hydrophobic Amino Acids

N alpha-Acetylenkephalin carboxypeptidase was co-purified with N-acetyltyrosine deacetylase from monkey kidney. Almost 90% of the activity from the homogenate was recovered in a high-speed supernatant without the use of detergents. The crucial steps in the purification were Cibacron Blue F3GA-Sepharose chromatography (involving negative and positive binding sequentially) and metal chelate affinity chromatography. The purified enzyme showed three bands on gel electrophoresis under non-denaturing conditions. All the three bands exhibited both N-acetyltyrosine deacetylase and N-acetylenkephalin carboxypeptidase activity, indicating their co-migration, Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence and absence of 2-mercaptoethanol gave a single protein band of mol.wt. 34 000. The native enzyme was a dimer of mol.wt. 66 000 as observed on Bio-Gel P-300 gel filtration. The carboxypeptidase removed two amino acids from the C-terminal end of either N-acetyl[Met5]- or N-acetyl[Leu5]-enkephalin. Non-acetylated enkephalins were less active as substrates. Peptides with their carboxy end blocked were inactive as substrates. Models suggested for carboxypeptidase A [Hartsuck & Lipscomb (1971) Enzymes 3, 1-56] support the idea that the kidney N-acetylated aromatic amino acid deacetylase or acylase III [Endo (1978) Biochim. Biophys. Acta 523, 207-217] can act as a carboxypeptidase on peptides having hydrophobic amino acids at the C-terminal end.

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Molecular forms of myeloperoxidase in human plasma

Biochemical Journal ◽

10.1042/bj2370559 ◽

1986 ◽

Vol 237 (2) ◽

pp. 559-565 ◽

Cited By ~ 19

Author(s):

R L Olsen ◽

T K Steigen ◽

T Holm ◽

C Little

Keyword(s):

Ion Exchange ◽

Human Plasma ◽

Gel Electrophoresis ◽

Large Subunit ◽

Polyacrylamide Gel Electrophoresis ◽

Immunoaffinity Chromatography ◽

Molecular Forms ◽

Active Components ◽

Protein Blotting ◽

Catalytically Active

A radioimmunoassay for myeloperoxidase was established with the use of affinity-purified anti-(human myeloperoxidase) immunoglobulins. By the use of ion-exchange followed by immunoaffinity chromatography a preparation of immunoreactive, catalytically active myeloperoxidase was obtained from fresh human plasma. In non-denaturing gel electrophoresis, the plasma preparation showed about four catalytically active components of mobility very similar to that of the granulocyte enzyme. SDS/polyacrylamide-gel electrophoresis combined with protein blotting showed that the two polypeptides of strongest antigenicity in the plasma preparation corresponded in Mr to the large and the small subunits of the granulocyte enzyme. In addition, the plasma preparation contained a higher-Mr immunoreactive polypeptide, possibly a precursor form of the enzyme, together with another of Mr similar to that of the large subunit of eosinophil peroxidase.

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