Purification of a third distinct xylanase from the xylanolytic system of Trichoderma harzianum

1986 ◽  
Vol 32 (7) ◽  
pp. 570-576 ◽  
Author(s):  
Ken K. Y. Wong ◽  
Larry U. L. Tan ◽  
John N. Saddler ◽  
Makoto Yaguchi
Keyword(s):  
Molecular Mass ◽  
Trichoderma Harzianum ◽  
Thermal Tolerance ◽  
Specific Activity ◽  
Gene Products ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Polysulfone Membrane ◽  
Xylan Hydrolysis ◽  
Hydrolysis Of

Three of the xylanases produced by Trichoderma harzianum E58 passed through a polysulfone membrane with molecular mass cut-off of 10 000 daltons, even though their molecular mass had been estimated to be 20 000, 22 000, and 29 000 daltons. The 22 000 dalton xylanase was purified to homogeneity from a preparation containing a mixture of 22 000 and 20 000 dalton xylanase using a combination of hydrophobic column chromatography and chromatofocusing. This enzyme has a pI of 8.5, a specific activity of 0.28 U/mg, a temperature optimum between 45 and 50 °C, a pH optimum between 4.5 and 5.0, and the ability to cleave xylotriose. It differs from the other two xylanases by having a lower pI, a lower specific activity, and a lower thermal tolerance. All three xylanases are highly specific for xylan hydrolysis and they do not cleave xylobiose or release arabinose substituents from arabinoxylan. Their amino acid compositions suggest that they are three distinct gene products. The three enzymes are major components of the xylanolytic system of T. harzianum, which consists of at least two other xylanases and two β-xylosidases which are responsible for the release of arabinose substituents and the hydrolysis of xylobiose.

scholarly journals Pereparation and characterization of two types of covalently immobilized amyloglucosidase

2005 ◽  
Vol 70 (5) ◽  
pp. 713-719 ◽  
Author(s):  
Nenad Milosavic ◽  
Radivoje Prodanovic ◽  
Slobodan Jovanovic ◽  
Irena Novakovic ◽  
Zoran Vujcic
Keyword(s):  
Specific Activity ◽  
Periodate Oxidation ◽  
Immobilized Enzymes ◽  
Soluble Starch ◽  
Soluble Enzyme ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Higher Temperature ◽  
Hydrolysis Of

Amyloglucosidase from A. niger was covalently immobilized onto poly( GMA-co-EGDMA) by the glutaraldehyde and periodate method. The immobilization of amyloglucosidase after periodate oxidation gave a preparate with the highest specific activity reported so far on similar polymers. The obtained immobilized preparates show the same pH optimum, but a higher temperature optimum compared with the soluble enzyme. The kinetic parameters for the hydrolysis of soluble starch by free and both immobilized enzymes were determined. .

AN ISOPHENOXAZINE SYNTHASE FROM PYGNOPORUS COCCINEUS (FR.) BOND. AND SING.

1964 ◽  
Vol 42 (11) ◽  
pp. 1515-1526 ◽  
Author(s):  
P. M. Nair ◽  
L. C. Vining
Keyword(s):  
Electron Acceptor ◽  
Specific Activity ◽  
Fold Increase ◽  
Maximum Activity ◽  
Flavin Mononucleotide ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Hydroxyanthranilic Acid ◽  
Pycnoporus Coccineus ◽  
Citrate Phosphate

Mycelium from cultures of the red polypore Pycnoporus coccineus (Fr.) Bond. and Sing, contains an enzyme which catalyzes the oxidative condensation of 2 molecules of 2-aminophenol to yield 2-amino-3H-isophenoxazin-3-one. Fractionation of the crude extract has given a preparation with an 893-fold increase in specific activity. The purified enzyme has a pH optimum in citrate-phosphate buffer of 5.0, and a temperature optimum of 55°. The Km value is 4.35 × 10−4 M. FMN and Mn++ ions were required for maximum activity. FAD also served as an electron acceptor. Of the metal ions tested only Mn++ activated the reaction. Hg++ and Fe++ inhibited strongly. The course of the reaction when cofactors were added separately suggested that flavin mononucleotide is the initial electron acceptor and that Mn++ ions are required for reoxidation of the flavin. The enzyme has narrow specificity, and does not catalyze the oxidation of 3-hydroxyanthranilic acid, 3-hydroxykynurenine, or pyrocatechol.

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.

scholarly journals Purification and properties of a 1,3-1,4-β-d-glucanase (lichenase, 1,3-1,4-β-d-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli

1988 ◽  
Vol 255 (3) ◽  
pp. 833-841 ◽  
Author(s):  
J D Erfle ◽  
R M Teather ◽  
P J Wood ◽  
J E Irvin
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Temperature Optimum ◽  
Glucanase Activity ◽  
Ph Optimum ◽  
Beta Glucanase ◽  
Purification And Properties ◽  
Hydrolysis Of

A 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli was purified 600-fold by chromatography on Q-Sepharose and hydroxyapatite. The cloned enzyme hydrolysed lichenin and oat beta-D-glucan but not starch, CM(carboxymethyl)-cellulose, CM-pachyman, laminarin or xylan. The enzyme had a broad pH optimum with maximum activity at approx. pH 6.0 and a temperature optimum of 50 degrees C. The pH of elution from a chromatofocusing column for the cloned enzyme was 4.7 (purified) and 4.9 (crude) compared with 4.8 for the mixed-linkage beta-D-glucanase activity in B. succinogenes. The Mr of the cloned enzyme was estimated to be 37,200 by gel filtration and 35,200 by electrophoresis. The Km values estimated for lichenin and oat beta-D-glucan were 0.35 and 0.71 mg/ml respectively. The major hydrolytic products with lichenin as substrate were a trisaccharide (82%) and a pentasaccharide (9.5%). Hydrolysis of oat beta-D-glucan yielded a trisaccharide (63.5%) and a tetrasaccharide (29.6%) as the major products. The chromatographic patterns of the products from the cloned enzyme appear to be similar to those reported for the mixed-linkage beta-D-glucanase isolated from Bacillus subtilis. The data presented illustrate the similarity in properties of the cloned mixed-linkage enzyme and the 1,3-1,4-beta-D-glucanase from B. subtilis and the similarity with the 1,4-beta-glucanase in B. succinogenes.

Properties of phospholipase A2 isolated from rat serum

1991 ◽  
Vol 69 (5-6) ◽  
pp. 358-365 ◽  
Author(s):  
R. R. Baker ◽  
H.-y. Chang
Keyword(s):  
Fatty Acid ◽  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Phospholipase A ◽  
Purification Step ◽  
Ph Optimum ◽  
Rat Serum ◽  
Absolute Requirement ◽  
Hydrolysis Of

Phospholipase A2 was extensively purified (1300- to 1400-fold) from rat serum using Sephadex G-100 chromatography. It eluted at a position corresponding to a molecular mass of about 15 kDa. This one purification step gave two bands on sodium dodecyl sulfate – polyacrylamide gel electrophoresis. The faster component had a molecular mass of 16 kDa and the slower band likely contained an aggregate of the faster component. Activity was associated with protein bands on nondenaturing gels. Enzyme activity was assessed using phosphatidylcholine or phosphatidylethanol-amine labelled at sn position 2 with radioactive arachidonate. Phosphatidylethanolamine gave higher specific activities than phosphatidylcholine. The enzyme has an absolute requirement for Ca2+ and a pH optimum at 7.4. This pH optimum was more prominent for phosphatidylethanolamine. Activity was inhibited by oleate or arachidonate when phosphatidylcholine was used as substrate, but added free fatty acid did not significantly affect the hydrolysis of phosphatidylethanolamine. Addition of bovine serum albumin (fatty acid free) to assays increased the rate of release of arachidonate from phosphatidylcholine, but not from phosphatidylethanolamine. Phospholipase A2 is present in serum likely as a consequence of blood coagulation and may release fatty acids from cellular membranes following hemorrhage.Key words: phospholipase A2, phosphatidylcholine, phosphatidylethanolamine, rat serum.

A novel ribonuclease from the veiled lady mushroom Dictyophora indusiata

2003 ◽  
Vol 81 (6) ◽  
pp. 373-377 ◽  
Author(s):  
Hexing Wang ◽  
Tzi Bun Ng
Keyword(s):  
Molecular Mass ◽  
Deae Cellulose ◽  
Transfer Rna ◽  
Rnase Activity ◽  
Fruiting Bodies ◽  
Terminal Sequence ◽  
Temperature Optimum ◽  
Ph Optimum

A ribonuclease (RNase), exhibiting a molecular mass of 28 kDa and specificity toward polyU and polyA and possessing an N-terminal sequence dissimilar to previously reported mushroom RNases, was isolated from dried fruiting bodies of veiled lady mushroom (Dictyophora indusiata). It demonstrated an RNase activity of 564 U/mg toward yeast transfer RNA. The RNase was adsorbed on DEAE-cellulose, CM-Sepharose, and Q-Sepharose. It demonstrated a pH optimum of 4–4.5 and a temperature optimum of 60 °C. There was a loss of RNase activity at temperatures above 60 °C.Key words: ribonuclease, Dictyophora indusiata, mushroom, purification.

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.

scholarly journals Preparation and Characterization of 5′-Phosphodiesterase from Barley Malt Rootlets

2010 ◽  
Vol 5 (2) ◽  
pp. 1934578X1000500
Author(s):  
Jie Hua ◽  
Ke-long Huang
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Barley Malt

Two 5′- phosphodiesterases (5′-PDE-a and 5′-PDE-b) were isolated from barley malt rootlets, and further purified by gel filtration on Sephadex G-25 and Sephadex G-75. 5′-PDE-a had a pH optimum of 5.0, temperature optimum of 70oC, and specific activity of 0.0143 mM ·mg−1-min−1. 5′-PDE –b had a pH optimum of 6.0, temperature optimum of 65°C and specific activity of 0.0125 mM ·mg−1·min−1. Both enzymes can be used to hydrolyze RNA to form 5′-nucleotides. The enzymes were quite stable at 70oC for 420 minutes. The Km was 0.24 mM for 5′-PDE-a and 0.16 mM for 5′-PDE-b with t-RNA (yeast) as substrate.

Enzymic hydrolysis of the potassium chloride soluble fraction of carrageenan: properties of "λ-carrageenases" from Pseudomonas carrageenovora

1973 ◽  
Vol 19 (7) ◽  
pp. 779-788 ◽  
Author(s):  
K. H. Johnston ◽  
E. L. McCandless
Keyword(s):  
Soluble Fraction ◽  
Reducing Sugar ◽  
Chondrus Crispus ◽  
Enzymic Hydrolysis ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Specific Viscosity ◽  
Chromatographic Resolution ◽  
Fraction I ◽  
Hydrolysis Of

An enzyme complex which hydrolyzed the KCl soluble carrageenan extracted from the red alga Chondrus crispus has been isolated from the cell-free medium of a culture of Pseudomonas carrageenovora grown on this polysaccharide. Three hydrolases could be separated. Fraction I, which caused a rapid decrease in the specific viscosity of the polysaccharide preparation with only minimal release of reducing sugar, could be distinguished from fraction II chromatographically on Sephadex G-100 and electrophoretically on agarose gel. Fraction IIa caused release of reducing sugar at pH 6.2, which activity was depressed at pH 7.5. Fraction IIb exhibited viscometric activity only at both pH 6.2 and pH 7.5. Fraction IIa had a sharp pH optimum at pH 6.2 and a temperature optimum at 28°. All hydrolases were inactivated by freezing, by dialysis against distilled water, by heating at 35° for 30 min, and by Hg2+ and 0.0001 mM EDTA. When fraction II (a and b) isolated after chromatographic resolution on Sephadex G-100 was incubated at pH 6.2 with KCl soluble carrageenan from C. crispus, products which had Rgal values of 0.74 and 0.17 were detected, were sulfated, and contained no 3,6-anhydrogalactose.

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.

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