Purification of the major endoglucanase from Aspergillus fumigatus Fresenius.
ABSTRACT: Aspergillus fumigatus (Fresenius), IMI 246651, A.T.C.C. 46324, produces two beta-glucosidase enzymes, cotton-solubilizing activity, xylanase and endoglucanase enzymes which can be separated by gel-filtration chromatography. The major endoglucanase does not bind to concanavalin A-Sepharose and does not stain with periodic acid/Schiff reagent. It is homogeneous on polyacrylamide isoelectric focusing (pI = 7.1) and has a mol.wt. of 12500 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The endoglucanase produces glucose and a mixture of oligosaccharides from cellulose; the purified enzyme has a small dextranase activity. It is stable at 50 degrees C and pH 6.
Project description:An endoglucanase (1,4-beta-D-glucan glucanohydrolase, EC 22.214.171.124) was purified from Clostridium thermocellum by procedures that included centrifugation, ultrafiltration, selective precipitation, ion-exchange Sephadex chromatography and preparative gel electrophoresis. The 22-fold-purified enzyme behaved as a homogeneous protein under non-denaturing conditions. The enzyme represented a significant component (greater than 25%) of total extracellular endoglucanase activity, but was purified in low yield by the procedures employed. The native molecular weight of the endoglucanase was determined by ultracentrifugational analysis, amino acid composition and polyacrylamide-gel electrophoresis, and varied between 83000 and 94000. The enzyme contained 11.2% carbohydrate and was isoelectric at pH 6.72. The pH and temperature optima of the endoglucanase were 5.2 and 62 degrees C respectively. The enzyme lacked cysteine and was low in sulphur-containing amino acids. The purified endoglucanase displayed: high activity towards carboxymethylcellulose, celloheptaose, cellohexaose and cellopentaose; low activity towards Avicel microcrystalline cellulose and cellotetraose; no detectable activity towards cellotriose or cellobiose; increased activity towards cello-oligosaccharides with increasing degree of polymerization. The internal glycosidic bonds of cello-oligosaccharides were cleaved by the enzyme in preference to external linkages. The apparent Michaelis constant ([S]0.5V) and Vmax. for cellopentaose and cellohexaose hydrolysis were 2.30 mM and 39.3 mumol/min per mg of protein, and 0.56 mM and 58.7 mumol/min per mg of protein, respectively.
Project description:A multifunctional transgenic Lactobacillus with probiotic characteristics and an ability to degrade ?-glucan and phytic acid (phytate) was engineered to improve nutrient utilization, increase production performance and decrease digestive diseases in broiler chickens. The Bacillus subtilis WL001 endoglucanase gene (celW) and Aspergillus fumigatus WL002 phytase gene (phyW) mature peptide (phyWM) were cloned into an expression vector with the lactate dehydrogenase promoter of Lactobacillus casei and the secretion signal peptide of the Lactococcus lactis usp45 gene. This construct was then transformed into Lactobacillus reuteri XC1 that had been isolated from the gastrointestinal tract of broilers. Heterologous enzyme production and feed effectiveness of this genetically modified L. reuteri strain were investigated and evaluated. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that the molecular mass of phyWM and celW was approximately 48.2 and 55 kDa, respectively, consistent with their predicted molecular weights. Endoglucanase and phytase activities in the extracellular fraction of the transformed L. reuteri culture were 0.68 and 0.42 U/mL, respectively. Transformed L. reuteri improved the feed conversion ratio of broilers from 21 to 42 days of age and over the whole feeding period. However, there was no effect on body weight gain and feed intake of chicks. Transformed L. reuteri supplementation improved levels of ash, calcium and phosphorus in tibiae at day 21 and of phosphorus at day 42. In addition, populations of Escherichia coli, Veillonella spp. and Bacteroides vulgatus were decreased, while populations of Bifidobacterium genus and Lactobacillus spp. were increased in the cecum at day 21.
Project description:The nucleotide sequence of the celG gene of Clostridium thermocellum, encoding endoglucanase CelG, was determined. The open reading frame extended over 1,698 bp and encoded a 566-amino-acid polypeptide (molecular weight of 63,128) similar to the C. thermocellum endoglucanase CelB (51.5% identical residues). The N terminus displayed a typical signal peptide, followed by a catalytic domain. The C terminus, which was separated from the catalytic domain by a 25-amino-acid segment rich in Pro, Thr, and Ser, contained two conserved stretches of 22 amino acids closely similar to those previously described in other cellulases from the same organism. Expression of the gene in Escherichia coli was increased by fusing the fragment coding for the catalytic domain in frame with the start of the lacZ' gene present in the vector. A low- and a high-M(r) form of the protein were purified. The two forms displayed identical enzymatic properties. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that both forms consist of a major polypeptide of M(r) 50,000 and two minor polypeptides of M(r)s 49,000 and 48,000, resulting from heterogeneous proteolytic cleavage at the C terminus. An antiserum raised against the forms purified from E. coli reacted with an immunoreactive polypeptide of M(r) 66,000, which was associated with the extracellular cellulolytic complex of C. thermocellum known as the cellulosome.
Project description:Brown rot basidiomycetes have long been thought to lack the processive cellulases that release soluble sugars from crystalline cellulose. On the other hand, these fungi remove all of the cellulose, both crystalline and amorphous, from wood when they degrade it. To resolve this discrepancy, we grew Gloeophyllum trabeum on microcrystalline cellulose (Avicel) and purified the major glycosylhydrolases it produced. The most abundant extracellular enzymes in these cultures were a 42-kDa endoglucanase (Cel5A), a 39-kDa xylanase (Xyn10A), and a 28-kDa endoglucanase (Cel12A). Cel5A had significant Avicelase activity--4.5 nmol glucose equivalents released/min/mg protein. It is a processive endoglucanase, because it hydrolyzed Avicel to cellobiose as the major product while introducing only a small proportion of reducing sugars into the remaining, insoluble substrate. Therefore, since G. trabeum is already known to produce a beta-glucosidase, it is now clear that this brown rot fungus produces enzymes capable of yielding assimilable glucose from crystalline cellulose.
Project description:The isolation and properties of a group of alcohols from the mycelium of Aspergillus fumigatus Fresenius are described. Mass-, nuclear-magnetic-resonance- and infrared-spectrometric studies coupled with evidence from ozonolytic degradation and chromatography show the mixture to contain hexahydroprenols-18, -19, -20, -21, -22, -23 and -24. Each contains a saturated ;hydroxy-terminal' isoprene residue, a saturated omega-terminal isoprene residue and a saturated zeta-isoprene residue (adjacent to the omega-residue). The presence of only two trans-isoprene residues is also a feature of the series of alcohols, but the precise position of these in each molecule is not known.
Project description:"Caldocellum saccharolyticum" is an obligatory anaerobic thermophilic bacterium. A gene from this organism, designated celB, has been cloned in Escherichia coli as part of a bacteriophage lambda gene library. This gene produces a thermostable cellulase that shows both endoglucanase and exoglucanase activities on test substrates and is able to degrade crystalline cellulose to glucose. The sequence of celB has homology with both exo- and endoglucanases described by others. It appears to have a central domain without enzymatic activity which is joined to the enzymatic domains by runs of amino acids rich in proline and threonine (PT boxes). Deletion analysis shows that the exoglucanase activity is located in the amino-terminal domain of the enzyme and that endoglucanase activity is located in the carboxy-terminal domain. There are internal transcriptional and translational start sites within the gene. The intact gene has been cloned into a temperature-inducible expression vector, pJLA602, and overexpressed in E. coli. Polyacrylamide gel electrophoresis showed that celB produced a protein with a molecular weight of 118,000 to 120,000. A number of smaller proteins with activity against carboxymethyl cellulose and 4-methyl umbelliferyl-beta-D-cellobioside were also produced. These are believed to be the result of alternative translational start sites and/or proteolytic degradation products of the translated gene product.
Project description:A strain of Aspergillus fumigatus Fresenius, isolated from spoiled hay, converts melilotic acid (o-hydroxyphenylpropionic acid) and o-coumaric acid into 4-hydroxycoumarin and dicoumarol. The sequence is shown to be melilotic acid (I) [Formula: see text] coumaric acid (IV) [Formula: see text] beta-hydroxymelilotic acid (II) [Formula: see text] beta-oxomelilotic acid (III) [Formula: see text] 4-hydroxycoumarin (VI), on the basis of (1) studies on the formation of postulated intermediates, (2) experiments with isotopically labelled materials and (3) sequential enzyme induction. In the presence of semicarbazide, o-coumaraldehyde is formed from o-coumaric acid: there is no evidence, however, that this lies on the normal metabolic pathway.
Project description:The rumen anaerobic cellulolytic bacterium Eubacterium cellulosolvens produces a large range of cellulases and hemicellulases responsible for the efficient hydrolysis of plant cell wall polysaccharides. One of these enzymes, endoglucanase Cel5A, comprises a tandemly repeated carbohydrate-binding module (CBM65) fused to a glycoside hydrolase family 5 (Cel5A) catalytic domain, joined by flexible linker sequences. The second carbohydrate-binding module located at the C-terminus side of the endoglucanase (CBM65B) has been co-crystallized with either cellohexaose or xyloglucan heptasaccharide. The crystals belong to the hexagonal space group P6(5) and tetragonal space group P4(3)2(1)2, containing a single molecule in the asymmetric unit. The structures of CBM65B have been solved by molecular replacement.
Project description:An endoglucanase (1,4-beta-D-glucan glucanohydrolase, EC 126.96.36.199) from the thermophilic anaerobe Clostridium thermocellum was purified to apparent homogeneity without the use of denaturants. No carbohydrate is associated with the endoglucanase. A molecular mass of 76,000 Da was determined by SDS/PAGE. The optimal pH is 7.0 and the enzyme is isoelectric at pH 5.05. The enzyme has a temperature optimum of 70 degrees C and retains approx. 50% of its activity after 48 h at 60 degrees C. Hydrolysis of CM-cellulose takes place with a rapid decrease in viscosity but a slow liberation of reducing sugars, indicating an endoglucanase type of activity. The endoglucanase shows little ability to hydrolyse highly ordered cellulose. Cellobiose inhibits whereas Mg2+ and Ca2+ stimulate the activity. The enzyme is completely inactivated by 1 mM-Hg2+ and is inhibited by a thiol-blocking reagent.
Project description:The deduced amino acid sequence derived from a Macrophomina phaseolina beta-1,4-endoglucanase-encoding gene revealed 48% identity (over 119 amino acids) with egl1 from the phytopathogen Pseudomonas solanacearum. Its similarity to saprophyte endoglucanases was not significant. Its minimum substrate size, unlike that of any known saprophyte endoglucanase, was cellopentaose. The unique characteristics of M. phaseolina egl1-encoded endoglucanase suggest that it is phytopathogen specific.