Purification and characterization of an endo-β-1,3-glucanase from Trichoderma harzianum

1996 ◽  
Vol 42 (10) ◽  
pp. 1039-1044 ◽  
Author(s):  
Eliane Ferreira Noronha ◽  
Cirano José Ulhoa
Keyword(s):  
Trichoderma Harzianum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Hydrophobic Interaction Chromatography ◽  
Maximum Activity ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Isolated Cell

β-1,3-Glucanases are produced by Trichoderma harzianum when it is grown in the presence of chitin or isolated cell wall from fungi. An endo-β-1,3-glucanase from the culture filtrate of T. harzianum was purified by gel filtration on Sephacryl S-200, followed by hydrophobic interaction chromatography on phenyl-Sepharose. A typical procedure provided 134-fold purification with a 3.6% yield. The molecular mass of the purified endo-β-1,3-glucanase was found to be approximately 36 kDa, as estimated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis on a 10% w/v slab gel. The enzyme was active toward glucans containing β-1,3-linkages and hydrolysed laminarin to form oligosaccharides. The Km and Vmax values for β-1,3-ghicanases, using laminarin as substrate, was 1.18 mg∙mL−1 and 1.26 U∙mL−1, respectively. The pH optimum for the enzyme was pH 4.4 and maximum activity was obtained at 45–50 °C. Enzyme activity was strongly inhibited in the presence of HgCl2 and stimulated by cations such as Zn2+ and Ca2+.Key words: endo-β-1,3-glucanase, Trichoderma harzianum, purification, characterization.

Purification and Characterization of an Acetyl Xylan Esterase from Bacillus pumilus

1998 ◽  
Vol 64 (2) ◽  
pp. 789-792 ◽  
Author(s):  
Giuliano Degrassi ◽  
Benedict C. Okeke ◽  
Carlo V. Bruschi ◽  
Vittorio Venturi
Keyword(s):  
Gel Electrophoresis ◽  
Bacillus Pumilus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Acetyl Xylan Esterase ◽  
Purification And Characterization ◽  
Michaelis Constant ◽  
Naphthyl Acetate

ABSTRACT Bacillus pumilus PS213 was found to be able to release acetate from acetylated xylan. The enzyme catalyzing this reaction has been purified to homogeneity and characterized. The enzyme was secreted, and its production was induced by corncob powder and xylan. Its molecular mass, as determined by gel filtration, is 190 kDa, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of 40 kDa. The isoelectric point was found to be 4.8, and the enzyme activity was optimal at 55°C and pH 8.0. The activity was inhibited by most of the metal ions, while no enhancement was observed. The Michaelis constant (Km ) andV max for α-naphthyl acetate were 1.54 mM and 360 μmol min−1 mg of protein−1, respectively.

scholarly journals Purification and Characterization of 1-Naphthol-2-Hydroxylase from Carbaryl-Degrading Pseudomonas Strain C4

2007 ◽  
Vol 189 (7) ◽  
pp. 2660-2666 ◽  
Author(s):  
Vandana P. Swetha ◽  
Aditya Basu ◽  
Prashant S. Phale
Keyword(s):  
Molecular Mass ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Substrate Inhibition ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sole Source ◽  
Maximum Activity ◽  
Ph Optimum ◽  
Phenol Derivatives

ABSTRACT Pseudomonas sp. strain C4 metabolizes carbaryl (1-naphthyl-N-methylcarbamate) as the sole source of carbon and energy via 1-naphthol, 1,2-dihydroxynaphthalene, and gentisate. 1-Naphthol-2-hydroxylase (1-NH) was purified 9.1-fold to homogeneity from Pseudomonas sp. strain C4. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme is a homodimer with a native molecular mass of 130 kDa and a subunit molecular mass of 66 kDa. The enzyme was yellow, with absorption maxima at 274, 375, and 445 nm, indicating a flavoprotein. High-performance liquid chromatography analysis of the flavin moiety extracted from 1-NH suggested the presence of flavin adenine dinucleotide (FAD). Based on the spectral properties and the molar extinction coefficient, it was determined that the enzyme contained 1.07 mol of FAD per mol of enzyme. Although the enzyme accepts electrons from NADH, it showed maximum activity with NADPH and had a pH optimum of 8.0. The kinetic constants Km and V max for 1-naphthol and NADPH were determined to be 9.6 and 34.2 μM and 9.5 and 5.1 μmol min−1 mg−1, respectively. At a higher concentration of 1-naphthol, the enzyme showed less activity, indicating substrate inhibition. The Ki for 1-naphthol was determined to be 79.8 μM. The enzyme showed maximum activity with 1-naphthol compared to 4-chloro-1-naphthol (62%) and 5-amino-1-naphthol (54%). However, it failed to act on 2-naphthol, substituted naphthalenes, and phenol derivatives. The enzyme utilized one mole of oxygen per mole of NADPH. Thin-layer chromatographic analysis showed the conversion of 1-naphthol to 1,2-dihydroxynaphthalene under aerobic conditions, but under anaerobic conditions, the enzyme failed to hydroxylate 1-naphthol. These results suggest that 1-NH belongs to the FAD-containing external flavin mono-oxygenase group of the oxidoreductase class of proteins.

Purification and Characterization of Kallikrein from Plasma of Patients with Acute Pancreatitis

1981 ◽  
Vol 60 (2) ◽  
pp. 199-205 ◽  
Author(s):  
Naotika Toki ◽  
Hiroyuki Sumi ◽  
Sumiyoshi Takasugi
Keyword(s):  
Acute Pancreatitis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Purification And Characterization ◽  
Α2 Macroglobulin ◽  
Sepharose 4B

1. A kallikrein-like enzyme in plasma of patients with acute pancreatitis was further purified by successive hydroxyapatite/cellulose and Sepharose-4B column chromatography. 2. By these procedures 0.26 mg of purified enzyme with a specific activity of 215 S-2266 chromozyme units/mg of protein was obtained from 10 ml of original plasma. 3. The purified material was homogeneous as ascertained by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had an apparent molecular weight of 31 000 as measured by gel filtration on Sephadex G-200. 4. It was confirmed immunologically that this enzyme was pancreatic kallikrein, which is distinct from plasma kallikrein, and that it could combine with α2-macroglobulin only in the presence of trypsin.

scholarly journals Purification and Characterization of a Dimethoate-Degrading Enzyme of Aspergillus niger ZHY256, Isolated from Sewage

2001 ◽  
Vol 67 (8) ◽  
pp. 3746-3749 ◽  
Author(s):  
Yu-Huan Liu ◽  
Ying-Cheng Chung ◽  
Ya Xiong
Keyword(s):  
Aspergillus Niger ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Michaelis Constant ◽  
Degrading Enzyme

ABSTRACT A dimethoate-degrading enzyme from Aspergillus nigerZHY256 was purified to homogeneity with a specific activity of 227.6 U/mg of protein. The molecular mass of the purified enzyme was estimated to be 66 kDa by gel filtration and 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was found to be 5.4, and the enzyme activity was optimal at 50°C and pH 7.0. The activity was inhibited by most of the metal ions and reagents, while it was induced by Cu2+. The Michaelis constant (K m ) andV max for dimethoate were 1.25 mM and 292 μmol min−1 mg of protein−1, respectively.

scholarly journals Purification and properties of S-adenosylmethionine: aldoxime O-methyltransferase from Pseudomonas sp. N.C.I.B. 11652

1985 ◽  
Vol 226 (1) ◽  
pp. 147-153 ◽  
Author(s):  
D B Harper ◽  
J T Kennedy
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Maximum Activity ◽  
Polyacrylamide Gel ◽  
Pseudomonas Sp ◽  
Ph Optimum

An enzyme catalysing the O-methylation of isobutyraldoxime by S-adenosyl-L-methionine was isolated from Pseudomonas sp. N.C.I.B. 11652. The enzyme was purified 220-fold by DEAE-cellulose chromatography, (NH4)2SO4 fractionation, gel filtration on Sephadex G-100 and chromatography on calcium phosphate gel. Homogeneity of the enzyme preparation was confirmed by isoelectric focusing on polyacrylamide gel and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The enzyme showed a narrow pH optimum at 10.25, required thiol-protecting agents for activity and was rapidly denatured at temperatures above 35 degrees C. The Km values for isobutyraldoxime and S-adenosyl-L-methionine were respectively 0.24 mM and 0.15 mM. Studies on substrate specificity indicated that attack was mainly restricted to oximes of C4-C6 aldehydes, with preference being shown for those with branching in the 2- or 3-position. Ketoximes were not substrates for the enzyme. Gel filtration on Sephadex G-100 gave an Mr of 84 000 for the intact enzyme, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated an Mr of 37 500, suggesting the presence of two subunits in the intact enzyme. S-Adenosylhomocysteine was a powerful competitive inhibitor of S-adenosylmethionine, with a Ki of 0.027 mM. The enzyme was also susceptible to inhibition by thiol-blocking reagents and heavy-metal ions. Mg2+ was not required for maximum activity.

Purification and characterization of an extracellular exochitinase, β-N-acetylhexosaminidase, from the fungal mycoparasite Stachybotrys elegans

2002 ◽  
Vol 48 (4) ◽  
pp. 311-319 ◽  
Author(s):  
Greg Taylor ◽  
Suha Jabaji-Hare ◽  
Pierre M Charest ◽  
Wajahatullah Khan
Keyword(s):  
Rhizoctonia Solani ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Synthetic Medium ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Biochemical Properties ◽  
Purification And Characterization ◽  
Proteolytic Product

The mycoparasite Stachybotrys elegans produces two exo- and one endo-acting chitinases when grown on chitin. We purified to homogeneity one of the exo-acting chitinases, β-N-acetylhexosaminidase and partially characterized its physical and biochemical properties. The native enzyme has a molecular mass of 120 kDa when determined by gel filtration and 68 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis indicating that the native protein probably occurs as a dimer in solution. The purified β-N-acetylhexosaminidase is most active at pH 5.0 and 40°C and hydrolyzes the ρ-nitrophenyl-N-acetyl-β-D-glucosaminide with apparent Km of 84.6 µM. Polyclonal antibodies raised against the 68-kDa β-N-acetylhexosaminidase (NAG-68) indicated that the antibody is highly specific and recognizes the protein in crude filtrate preparation. This suggests that the protein is a not a proteolytic product of another protein. Western blot analysis showed that the activity of NAG-68 was induced when S. elegans was grown on purified cell wall fragments of its host, Rhizoctonia solani, as well as during antagonistic interaction of the mycoparasite and host when both were grown on synthetic medium with or without supplemental carbon source.Key words: chitinases, protein purification, mycoparsite, Rhizoctonia solani.

Carboxymethylhydroxymuconic semialdehyde dehydrogenase in the 4-hydroxyphenylacetate catabolic pathway of Pseudomonas putida

1986 ◽  
Vol 64 (12) ◽  
pp. 1288-1293 ◽  
Author(s):  
Josefa M. Alonso ◽  
Amando Garrido-Pertierra
Keyword(s):  
Pseudomonas Putida ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Competitive Inhibitor ◽  
Catabolic Pathway ◽  
Ph Optimum ◽  
Semialdehyde Dehydrogenase ◽  
Standard Assay ◽  
Assay Conditions

5-Carboxymethyl-2-hydroxymuconic semialdehyde (CHMSA) dehydrogenase in the 4-hydroxyphenylacetate meta-cleavage pathway was purified from Pseudomonas putida by gel filtration, anion-exchange, and affinity chromatographies. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis analysis suggested an approximate tetrameric molecular weight of 200 000. The purified enzyme showed a pH optimum at 7.8. The temperature–activity relationship for the enzyme from 27 to 45 °C showed broken Arrhenius plots with an inflexion at 36–37 °C. Under standard assay conditions, the enzyme acted preferentially with NAD. It could also catalyze the reduction with NADP (which had a higher Km), at 18% of the rate observed for NAD. The following kinetic parameters were found: Km(NAD) = 20.0 ± 3.6 μM, Km(CHMSA) = 8.5 ± 1.8 μM, and Kd(enzyme–NAD complex) = 7.8 ± 2.0 μM. The product NADH acted as a competitive inhibitor against NAD.

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

scholarly journals Biochemical and Genetic Characterization of an FK506-Sensitive Peptidyl Prolyl cis-trans Isomerase from a Thermophilic Archaeon, Methanococcus thermolithotrophicus

1998 ◽  
Vol 180 (2) ◽  
pp. 388-394 ◽  
Author(s):  
Masahiro Furutani ◽  
Toshii Iida ◽  
Shigeyuki Yamano ◽  
Kei Kamino ◽  
Tadashi Maruyama
Keyword(s):  
Amino Acid ◽  
Genomic Library ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ppiase Activity ◽  
Methanococcus Thermolithotrophicus ◽  
Fk506 Binding Protein ◽  
Thermophilic Archaeon

ABSTRACT A peptidyl prolyl cis-trans isomerase (PPIase) was purified from a thermophilic methanogen, Methanococcus thermolithotrophicus. The PPIase activity was inhibited by FK506 but not by cyclosporine. The molecular mass of the purified enzyme was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 42 kDa by gel filtration. The enzyme was thermostable, with the half-lives of its activity at 90 and 100°C being 90 and 30 min, respectively. The catalytic efficiencies (k cat/Km ) measured at 15°C for the peptidyl substrates,N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide andN-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, were 0.35 and 0.20 μM−1 s−1, respectively, in chymotrypsin-coupled assays. The purified enzyme was sensitive to FK506 and therefore was called MTFK (M. thermolithotrophicusFK506-binding protein). The MTFK gene (462 bp) was cloned from anM. thermolithotrophicus genomic library. The comparison of the amino acid sequence of MTFK with those of other FK506-binding PPIases revealed that MTFK has a 13-amino-acid insertion in the N-terminal region that is unique to thermophilic archaea. The relationship between the thermostable nature of MTFK and its structure is discussed.

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