A novel tannase from Aspergillus niger with β-glucosidase activity

Microbiology ◽  
2003 ◽  
Vol 149 (10) ◽  
pp. 2941-2946 ◽  
Author(s):  
M. Ascención Ramírez-Coronel ◽  
Gustavo Viniegra-González ◽  
Alan Darvill ◽  
Christopher Augur
Keyword(s):  
Aspergillus Niger ◽  
Hplc Analysis ◽  
Gel Filtration ◽  
Culture Broth ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Glucosidase Activity ◽  
Sds Page ◽  
Hydrolysable Tannins ◽  
Molecular Masses

An extracellular tannase was produced from solid-state cultures of Aspergillus niger. The enzyme was purified to homogeneity from the cell-free culture broth by preparative isoelectric focusing and by FPLC using anion-exchange and gel-filtration chromatography. SDS-PAGE analysis as well as gel localization studies of purified tannase indicated the presence of two enzyme forms, with molecular masses of 90 kDa and 180 kDa. The tannase had an isoelectric point of 3·8, a temperature optimum of 60–70 °C and a pH optimum of 6·0. The substrate specificity of the tannase was determined by HPLC analysis of tannin substrates and products. The enzyme was able to remove gallic acid from both condensed and hydrolysable tannins. Internal sequences were obtained from each of the gel-purified and trypsin-digested tannase forms. The peptide sequences obtained from both forms were identical to sequences within a β-glucosidase from Aspergillus kawachii. The purified tannase was tested for β-glucosidase activity and was shown to hydrolyse cellobiose efficiently. However, no β-glucosidase activity was detected when the enzyme was assayed in the presence of tannic acid.

scholarly journals Purification and characterization of three extracellular (1→3)-β-d-glucan glucohydrolases from the filamentous fungus Acremonium persicinum

1995 ◽  
Vol 308 (3) ◽  
pp. 733-741 ◽  
Author(s):  
S M Pitson ◽  
R J Seviour ◽  
B M McDougall ◽  
J R Woodward ◽  
B A Stone
Keyword(s):  
Filamentous Fungus ◽  
Gel Filtration ◽  
High Specificity ◽  
Major Product ◽  
Ph Optimum ◽  
Sds Page ◽  
Molecular Masses ◽  
Monomeric Proteins ◽  
Ph Range

Three (1-->3)-beta-D-glucanases (GNs) were isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. Homogeneity of the purified proteins was confirmed by SDS/PAGE, isoelectric focusing and N-terminal amino acid sequencing. All three GNs (GN I, II and III) are non-glycosylated, monomeric proteins with apparent molecular masses, estimated by SDS/PAGE, of 81, 85 and 89 kDa respectively. pI values for the three enzymes are 5.3, 5.1, and 4.4 respectively. The pH optimum for GN I is 6.5, and 5.0 for GN II and III. All three purified enzymes displayed stability over the pH range 4.5-10.0. Optimum activities for GN I, II and III were recorded at 65, 55 and 60 degrees C respectively, with both GN II and III having short-term stability up to 50 degrees C and GN I up to 55 degrees C. The purified GNs have high specificity for (1-->3)-beta-linkages and hydrolysed a range of (1-->3)-beta- and (1-->3)(1-->6)-beta-D-glucans, with laminarin from Laminaria digitata being the most rapidly hydrolysed substrate of those tested. K(m) values for GN I, II, and III against L. digitata laminarin were 0.1, 0.23 and 0.22 mg/ml respectively. D-Glucono-1,5-lactone does not inhibit any of the three GNs, some metals ions are mild inhibitors, and N-bromosuccinimide and KMnO4 are strong inhibitors. All three GNs acted in an exo-hydrolytic manner, determined by the release of alpha-glucose as the initial and major product of hydrolysis of (1-->3)-beta-D-glucans, and confirmed by viscometric analysis and the inability to cleave periodate-oxidized laminarin, and may be classified as (1-->3)-beta-D-glucan glucohydrolases (EC 3.2.1.58).

scholarly journals Purification and some characteristics of the acetylxylan esterase from Schizophyllum commune

1994 ◽  
Vol 298 (3) ◽  
pp. 751-755 ◽  
Author(s):  
N Halgasová ◽  
E Kutejová ◽  
J Timko
Keyword(s):  
Enzyme Activity ◽  
Ion Exchange ◽  
Molecular Mass ◽  
Schizophyllum Commune ◽  
Gel Filtration ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Full Activity ◽  
Sds Page ◽  
Acetylxylan Esterase

Acetylxylan esterase from Schizophyllum commune was purified using ion-exchange and hydrophobic chromatography. The enzyme has a molecular mass of 31 kDa, as determined by SDS/PAGE, or 18 kDa, according to gel filtration. Glycosylation of the enzyme was not detected. Acetylxylan esterase is relatively stable under laboratory conditions; it retains full activity at pH 6.2-8.5 upon incubation at 25 degrees C for 7 h, but loses nearly the whole activity upon incubation at 60 degrees C for 30 min. The pH optimum of the enzyme activity is 7.7 and its temperature optimum lies between 30 and 45 degrees C. Ca2+ and Co2+ inhibit markedly the activity of acetylxylan esterase at a concentration of 10 mM, as do Mn2+, Zn2+, Fe2+ and Cu2+ at a concentration of 1 mM.

Production of Geotrichum candidum polygalacturonases via solid state fermentation on grape pomace

2012 ◽  
Vol 66 (9) ◽  
Author(s):  
Kateřina Illková ◽  
Zuzana Zemková ◽  
Dana Flodrová ◽  
Jakub Jäger ◽  
Dagmar Benkovská ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Mass ◽  
Extracellular Enzymes ◽  
Gel Filtration ◽  
Pectin Methylesterase ◽  
Geotrichum Candidum ◽  
Grape Pomace ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Sds Page

AbstractGeotrichum candidum CCY 16-1-29 (teleomorph Galactomyces geotrichum) is able to grow and produce polygalacturonase of remarkable activities on pectin or grape pomace as a sole carbon source. The highest activities of extracellular enzymes were found on the third and the seventh day of cultivation. After extraction and precipitation, polygalacturonases produced in these cultivation periods were characterized. Production of multiple forms of polygalacturonase was observed in both cultivation periods. Two major forms, polygalacturonase with random action pattern (endo-PGase, EC 3.2.1.15) and oligogalacturonate hydrolase (exoPGase, exopolygalacturonase preferring oligogalacturonides as substrates), as well as numerous minor forms were detected by IEF-PAGE using the print technique detection. EndoPGase was identified by mass spectrometry. The major forms have similar isoelectric points (below pH 6.0) and pH optima (4.6 and 4.8, respectively). pH optimum of 4.6 was associated with exoPGase and that of 4.8 with endoPGase. Both enzymes were stable after freeze-drying and storage at 4°C. EndoPGase had molecular mass of about 29 kDa (36 kDa by SDS-PAGE) as determined by gel filtration, temperature optimum of about 45°C and it was stable only below 35°C. Molecular mass of exoPGase was about 50 kDa, its temperature optimum was about 60°C, and it was stable to 60°C. Optimal substrate for exoPGase was a pentamer, for endoPGase it was a pectate. Values of K m for optimal substrate reached the values of 11.4 × 10−5 M for for exoPGase and 6.6 × 10−5 M for endoPGase. Pectin methylesterase as another pectolytic enzyme was also identified by mass spectrometry.

scholarly journals Isolation and partial characterization of protease from Pseudomonas aeruginosa ATCC 27853

2010 ◽  
Vol 75 (8) ◽  
pp. 1041-1052 ◽  
Author(s):  
Lidija Izrael-Zivkovic ◽  
Gordana Gojgic-Cvijovic ◽  
Ivanka Karadzic
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Zinc Ion ◽  
Metal Chelators ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Sds Page ◽  
Potential Use

Enzymatic characteristics of a protease from medically important, referent strain of Pseudomonas aeruginosa ATCC 27853 were determined. According to SDS PAGE and gel filtration it was estimated that molecular mass of the purified enzyme was about 15 kDa. Other enzymatic properties were found to be: pH optimum 7.1, pH stability between pH 6.5 and pH 10; temperature optimum around 60?C while the enzyme was stable at 60?C for 30 min. The inhibition of the enzyme was observed with the metal chelators such as EDTA and 1,10- phenanthroline, suggesting that the protease is a metalloenzyme. Further more it was determined that enzyme contains one mole of zinc ion per mole of enzyme. The protease is stable in the presence of different organic solvents, which enable potential use for synthesis of peptides.

scholarly journals Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA

1997 ◽  
Vol 325 (3) ◽  
pp. 761-769 ◽  
Author(s):  
Isabelle GARCIA ◽  
Matthew RODGERS ◽  
Catherine LENNE ◽  
Anne ROLLAND ◽  
Alain SAILLAND ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Gel Filtration ◽  
Peptide Sequence ◽  
Amino Acid Residues ◽  
Carrot Cells ◽  
Expression Studies ◽  
Sds Page ◽  
Molecular Masses ◽  
The Difference ◽  
Dioxygenase Activity

p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45–46 kDa. This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens. Gel-filtration chromatography indicates that the enzyme behaves as a homodimer. We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase. The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases. This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells. Comparison of the N-terminal sequence of the 45–46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity. Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification. This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide.

scholarly journals Purification and characterization of cytosolic pyruvate kinase from banana fruit

2000 ◽  
Vol 352 (3) ◽  
pp. 875-882 ◽  
Author(s):  
William L. TURNER ◽  
William C. PLAXTON
Keyword(s):  
Pyruvate Kinase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Banana Fruit ◽  
Ph Optimum ◽  
Cytosolic Ph ◽  
Pep Carboxylase ◽  
Sds Page ◽  
Optimal Efficiency

Cytosolic pyruvate kinase (PKc) from ripened banana (Musa cavendishii L.) fruits has been purified 543-fold to electrophoretic homogeneity and a final specific activity of 59.7µmol of pyruvate produced/min per mg of protein. SDS/PAGE and gel-filtration FPLC of the final preparation indicated that this enzyme exists as a 240kDa homotetramer composed of subunits of 57kDa. Although the enzyme displayed a pH optimum of 6.9, optimal efficiency in substrate utilization [in terms of Vmax/Km for phosphoenolpyruvate (PEP) or ADP] was equivalent at pH6.9 and 7.5. PKc activity was absolutely dependent upon the presence of a bivalent and a univalent cation, with Mg2+ and K+ respectively fulfilling this requirement. Hyperbolic saturation kinetics were observed for the binding of PEP, ADP, Mg2+ and K+ (Km values of 0.098, 0.12, 0.27 and 0.91mM respectively). Although the enzyme utilized UDP, IDP, GDP and CDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate and MgATP were the most effective inhibitors, whereas L-aspartate functioned as an activator by reversing the inhibition of PKc by L-glutamate. The allosteric features of banana PKc are compared with those of banana PEP carboxylase [Law and Plaxton (1995) Biochem. J. 307, 807Ő816]. A model is presented which highlights the roles of cytosolic pH, MgATP, L-glutamate and L-aspartate in the co-ordinate control of the PEP branchpoint in ripening bananas.

Purification and characterization of the extracellular α-amylase activity of the yeast Schwanniomyces alluvius

1987 ◽  
Vol 65 (10) ◽  
pp. 899-908 ◽  
Author(s):  
F. Moranelli ◽  
M. Yaguchi ◽  
G. B. Calleja ◽  
A. Nasim
Keyword(s):  
Amino Acid ◽  
Amylase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Proteinase K ◽  
Ph Optimum ◽  
Sds Page

The extracellular α-amylase activity of the yeast Schwanniomyces alluvius has been purified by anion-exchange chromatography on DEAE-cellulose and gel-filtration chromatography on Sephadex G-100. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) and N-terminal amino acid analysis of the purified sample indicated that the enzyme preparation was homogeneous. The enzyme is a glycoprotein having a molecular mass of 52 kilodaltons (kDa) estimated by SDS–PAGE and 39 kDa by gel filtration on Sephadex G-100. Chromatofocusing shows that it is an acidic protein. It is resistant to trypsin but sensitive to proteinase K. Its activity is inhibited by the divalent cation chelators EDTA and EGTA and it is insensitive to sulfhydryl-blocking agents. Exogenous divalent cations are inhibitory as are high concentrations of monovalent salts. The enzyme has a pH optimum between 3.75 and 5.5 and displays maximum stability in the pH range of 4.0–7.0. Under the conditions tested, the activity is maximal between 45 and 50 °C and is very thermolabile. Analysis of its amino acid composition supports its acidic nature.

Identifying and characterizing the most significant β-glucosidase of the novel species Aspergillus saccharolyticus

2012 ◽  
Vol 58 (9) ◽  
pp. 1035-1046 ◽  
Author(s):  
Annette Sørensen ◽  
Birgitte K. Ahring ◽  
Mette Lübeck ◽  
Wimal Ubhayasekera ◽  
Kenneth S. Bruno ◽  
...  
Keyword(s):  
Hydrolytic Activity ◽  
Fungal Species ◽  
Culture Broth ◽  
Exchange Chromatography ◽  
Mass Spectrometry Analysis ◽  
Glycoside Hydrolase Family ◽  
Degenerate Primers ◽  
Spectrometry Analysis ◽  
Glucosidase Activity ◽  
Sds Page

The newly discovered fungal species Aspergillus saccharolyticus was found to produce a culture broth rich in β-glucosidase activity. In this present work, the main β-glucosidase of A. saccharolyticus responsible for the efficient hydrolytic activity was identified, isolated, and characterized. Ion exchange chromatography was used to fractionate the culture broth, yielding fractions with high β-glucosidase activity and only 1 visible band on an SDS–PAGE gel. Mass spectrometry analysis of this band gave peptide matches to β-glucosidases from aspergilli. Through a polymerase chain reaction approach using degenerate primers and genome walking, a 2919 bp sequence encoding the 860 amino acid BGL1 polypeptide was determined. BGL1 of A. saccharolyticus has 91% and 82% identity with BGL1 from Aspergillus aculeatus and BGL1 from Aspergillus niger , respectively, both belonging to Glycoside Hydrolase family 3. Homology modeling studies suggested β-glucosidase activity with preserved retaining mechanism and a wider catalytic pocket compared with other β-glucosidases. The bgl1 gene was heterologously expressed in Trichoderma reesei QM6a, purified, and characterized by enzyme kinetics studies. The enzyme can hydrolyze cellobiose, p-nitrophenyl-β-d-glucoside, and cellodextrins. The enzyme showed good thermostability, was stable at 50 °C, and at 60 °C it had a half-life of approximately 6 h.

scholarly journals Purification and characterization of an extracellular (1 → 6)-β-glucanase from the filamentous fungus Acremonium persicinum

1996 ◽  
Vol 316 (3) ◽  
pp. 841-846 ◽  
Author(s):  
Stuart M. PITSON ◽  
Robert J. SEVIOUR ◽  
Barbara M. McDOUGALL ◽  
Bruce A. STONE ◽  
Maruse SADEK
Keyword(s):  
Molecular Mass ◽  
Filamentous Fungus ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Eisenia Bicyclis ◽  
Ph Optimum ◽  
Hydrolysis Products ◽  
Sds Page ◽  
Hydrolysis Of

An endo-(1 → 6)-β-glucanase has been isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. SDS/PAGE of the purified enzyme gave a single band with an apparent molecular mass of 42.7 kDa. The enzyme is a non-glycosylated, monomeric protein with a pI of 4.9 and pH optimum of 5.0. It hydrolysed (1 → 6)-β-glucans (pustulan and lutean), initially yielding a series of (1 → 6)-β-linked oligoglucosides, consistent with endo-hydrolytic action. Final hydrolysis products from these substrates were gentiobiose and gentiotriose, with all products released as β-anomers, indicating that the enzyme acts with retention of configuration. The purified enzyme also hydrolysed Eisenia bicyclis laminarin, liberating glucose, gentiobiose, and a range of larger oligoglucosides, through the apparent hydrolysis of (1 → 6)-β- and some (1 → 3)-β-linkages in this substrate. Km values for pustulan, lutean and laminarin were 1.28, 1.38, and 1.67 mg/ml respectively. The enzyme was inhibited by N-acetylimidazole, N-bromosuccinimide, dicyclohexylcarbodi-imide, Woodward's Regent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4 and some metal ions, whereas D-glucono-1,5-lactone and EDTA had no effect.

A truncated β-xylosidase from the anaerobic fungus Neocallimastix patriciarum 27

1994 ◽  
Vol 40 (6) ◽  
pp. 484-490 ◽  
Author(s):  
Hong Zhu ◽  
K.-J. Cheng ◽  
Cecil W. Forsberg
Keyword(s):  
Ion Exchange ◽  
Peptide Mapping ◽  
Gel Filtration ◽  
Barley Straw ◽  
Anaerobic Fungus ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Neocallimastix Patriciarum ◽  
Molecular Masses ◽  
Temperature Optima

Two extracellular β-xylosidases, xylosidase I and II, were isolated from the ruminal fungus Neocallimastix patriciarum 27 after growth in a barley straw medium. Xylosidase I was purified 88-fold to apparent homogeneity by ion-exchange, affinity, and gel filtration chromatography. The purified xylosidase I had an isoelectric point (pI) of 4.7 and was a monomelic protein with a molecular mass of 39.5 kDa as estimated by both SDS-PAGE and gel filtration. Xylosidase II was partially purified to approximately 95% purity. Xylosidase II had the same pI (4.7) as xylosidase I, and appeared to be a dimeric enzyme composed of two polypeptides with molecular masses of 85 and 45 kDa, respectively, on SDS-PAGE. Peptide mapping of the three proteins suggested that xylosidase I was a truncated product originating from xylosidase II. Xylosidases I and II had similar pH optima of 6.0, but different temperature optima of 50 and 40 °C, respectively. The Km and Vmax for xylosidase I were 0.59 mM of p-nitrophenyl-β-D-xylopyranoside and 38.04 U∙mg protein−1, respectively, and those for xylosidase II were 0.13 mM and 8.9 U∙mg protein−1, respectively. Both enzymes hydrolysed pNPX and xylobiose with the production of xylose, but only xylosidase I exhibited activity toward p-nitrophenyl-α-L-arabinofuranoside.Key words: xylosidase, Neocallimastix, patriciarum, glycosidase.

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