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PubMedDate not supplied

[Detection of transglycosylation reactions under the influence of Trichoderma koningii and Trichoderma viride cellulase].

The transglycosylation reactions catalyzed by the low molecular weight endo-beta-1,4-glucanase from the cellulase complex of Trichoderma koningii as well as unseparated complexes from Tr. koningii and Tr. viride were investigated. The products obtained from the mixture of cellodextrine (donor, 0.5%) and p-nitrophenyl-beta-D-glucopyranoside (acceptor, 0.4%) with the aid of the above enzymes were analyzed. Transglycosylation products were separated by Sephadex G-15 gel-filtration. In all the three cases the highest molecular weight p-nitrophenyl glycosides of oligosaccharides (with a molecular weight of over 1500) were first synthesized and then hydrolyzed. At the beginning of the reaction the amount of p-nitrophenyl glycosides of oligosaccharides synthesized was greater than that of the reducing groups measured according to Nelson. Addition of p-nitrophenyl-beta-D-glucopyranoside to cellodextrine did not influence the rate with which the latter was hydrolyzed by endoglucanase.

Cellulase↗
PubMed1977

Kinetic properties and substrate specificities of two cellulases from auxin-treated pea epicotyls.

Two cellulases purified from growing regions of auxin-treated peas (buffer-soluble and buffer-insoluble) hydrolyze cellulose powder, partially substituted carboxymethylcellulose (CM-cellulose), higher cellodextrins, and certain mixed linkage glucans (e.g. barley beta-glucan), at rates comparable to these reported for the most active fungal cellulases, and with kinetics and product formation characteristic of endohydrolase action. They are unable to cleave 1,3-linkages in beta-glucans, or 1,4-linkages in dextrins containing excessive substitution at C6, alpha configuration, alternating beta-1,3- and 1,4-linkages, or residues other than anhydroglucose. They are not active towards cellobiose or the 1,4-linkage adjacent to the reducing end of cellodextrin chains. It is concluded that buffer-soluble and buffer-insoluble cellulases are true beta-1,4-glucan 4-glucanohydrolases (EC 3.2.1.4). On a molar basis, Vmax values for buffer-insoluble are higher than buffer-soluble cellulase acting towards any of the substrates tested, but Km values towards CM-cellulose and cellohexaose are essentially identical. Both cellulases were inhibited by C12+, Hg2+, and sulfhydryl-binding reagents. Buffer-insoluble, but not buffer-soluble, cellulose was inactivated by reagents that bind serine and threonine, which reflects differences in their amino acid composition. No major qualitative differences have been detected in the mode of action of the two enzymes. Despite marked differences in their physical and immunological properties, close similarities between buffer-soluble and buffer-insoluble enzymic properties suggest that their active sites are the same.

Cellulase↗
PubMed1990

Sequencing and expression of a cellodextrinase (ced1) gene from Butyrivibrio fibrisolvens H17c cloned in Escherichia coli.

The nucleotide sequence of a 2.314 kb DNA segment containing a gene (ced1) expressing cellodextrinase activity from Butyrivibrio fibrisolvens H17c was determined. The B. fibrisolvens H17c gene was expressed from a weak internal promoter in Escherichia coli and a putative consensus promoter sequence was identified upstream of a ribosome binding site and a GTG start codon. The complete amino acid sequence (547 residues) was deduced and homology was demonstrated with the Clostridium thermocellum endoglucanase D (EGD), Pseudomonas fluorescens var. cellulosa endoglucanase (EG), and a cellulase from the avocado fruit (Persea americana). The ced1 gene product Ced1 showed cellodextrinase activity and rapidly hydrolysed short-chain cellodextrins to yield either cellobiose or cellobiose and glucose as end products. The Ced1 enzyme released cellobiose from p-nitrophenyl-beta-D-cellobioside and the enzyme was not inhibited by methylcellulose, an inhibitor of endoglucanase activity. Although the major activity of the Ced1 enzyme was that of a cellodextrinase it also showed limited activity against endoglucanase specific substrates [carboxymethylcellulose (CMC), lichenan, laminarin and xylan]. Analysis by SDS-polyacrylamide gel electrophoresis with incorporated CMC showed a major activity band with an apparent Mr of approximately 61,000. The calculated Mr of the ced1 gene product was 61,023.

Amino Acid Sequence↗
PubMed1985

A cellulase assay coupled to cellobiose dehydrogenase.

An assay for cellulase activity based on the oxidation of cellobiose, formed during the cellulase reaction, with ferricyanide and a cellobiose dehydrogenase derived from the cellulolytic fungus Sporotrichum (Chrysosporium) thermophile is presented. Due to the restricted specificity of this enzyme for cellobiose and cellodextrins, glucose, which may be formed by the action of some cellulolytic components or by beta-glucosidase, does not contribute to the result. The negative interference of beta-glucosidase may be eliminated by glucono-delta-lactone inhibition. The assay, which is not influenced by cellobiose back-inhibition of the cellulase reaction, like the usual cellulase tests based on the increase in reducing power, is basically unspecific with respect to endo- or exo-acting enzymes giving rise to a total cellulase activity. With the use of an amorphous cellulose substrate (reprecipitated cellulose after dissolving in concentrated phosphoric acid), unpredictable effects due to cooperativity between endo- and exo-enzyme components were eliminated. An analytical procedure giving a linear response between activity and enzyme concentration and between activity and time of incubation has been worked out.

Carbohydrate Dehydrogenases↗
PubMed1992

Evidence that cellobiose:quinone oxidoreductase from Phanerochaete chrysosporium is a breakdown product of cellobiose oxidase.

Phanerochaete chrysosporium releases two enzymes that oxidize cellobiose and higher cellodextrins: the flavohaemoprotein cellobiose oxidase and the flavoprotein cellobiose:quinone oxidoreductase (CBQase). Partial digestion of these enzymes with Staphylococcal V8 proteinase or cyanogen bromide yielded many identical bands on SDS-polyacrylamide gels. A polyclonal antibody to either purified protein gave cross-reaction. The purification procedure also yielded a haem protein that ran on dodecyl sulphate gels at Mr 31,000, as compared with 91,000 for cellobiose oxidase and 63,000 for CBQase. The 31 kDa haem protein cross-reacted with polyclonal antibody to cellobiose oxidase, but not with antibody to CBQase. Sulphite bleached the flavin of cellobiose oxidase, but gave no reaction with the 31 kDa haem protein, suggesting an absence of flavin. It is proposed that CBQase and the 31 kDa haem protein are formed from cellobiose oxidase by proteolytic cleavage.

Agaricales↗
PubMed1995

Properties of a novel glucose-enhanced beta-glucosidase purified from Streptomyces sp. (ATCC 11238).

An inducible intracellular beta-glucosidase (EC 3.2.1.21) from Streptomyces sp. QM-B814 (ATCC 11238) has been purified and characterized. The purified polypeptide is monomeric with a relative molecular mass of 62 kDa by SDS-PAGE and 42 kDa by size-exclusion chromatography; its isoelectric point is 4.2. The difference in the molecular mass values can be attributed to the glycosylated nature of the protein. The purified enzyme has a pH optimum of 6.0-6.5. The temperature optimum for activity is 50 degrees C; at this temperature the enzyme is stable for 1 h. The enzyme hydrolyzes mainly aryl-beta-glucosides but also presents significant activity against beta-linked disaccharides and maltose. The enzyme displays an unusual kinetic behavior and biphasic Lineweaver-Burk and Eadie-Hofstee plots for p-nitrophenyl-beta-D-glucoside and cellobiose were obtained. The enzyme presents beta-glycosyltransferase activity and an exoglycosidase-type action on cellodextrins. It is inhibited by delta-gluconolactone (Ki 0.44 mM) but, remarkably, glucose in the range 25-200 mM enhances the rate of p-nitrophenyl-beta-D-glucoside hydrolysis.

Amino Acids↗
PubMed1975

Cellulose synthesis by Acetobacter xylinum. III. Matrix, primer and lipid requirements and heat stability of the cellulose-forming enzymes.

The addition of soluble cellodextrins of increasing size to a cell envelope preparation of Acetobacter xylinum stimulated cellulose synthesis from UDPG. This stimulation was attributed to both acceptor and activator effects. Enzymes required for cellulose synthesis were found to be heat-unstable and those required for synthesis of glycosylated lipid components from UDPG, heat-stable. Both heat-inactivated envelope fragments and supernatant fluid from whole cells were necessary for cellulose synthesis from UDPG. Cellulose was not formed from UDPG in the presence of either supernatant fluid alone or heat-inactivated envelopes alone. The combined results of this and previous studies suggest that either the cell envelope is necessary for synthesis of a more immediate precursor to cellulose than UDPG, or that the synthesis from UDPG requires a matrix. The former suggestion and its possible link with lipid intermediate involvement was strengthened by the observation of inefficient glycosylated lipid formation by a celluloseless mutant strain of A. xylinum. The possible locations of various enzyme activities required for the synthesis of the cellulose precursor are indicated and a possible microfibril nucleation process is discussed.

Cell Wall↗
PubMed1986

Purification and properties of the endoglucanase C of Clostridium thermocellum produced in Escherichia coli.

The celC gene, which codes for a new endoglucanase of Clostridium thermocellum, termed endoglucanase C, was found to be expressed when cloned in Escherichia coli. The enzyme was purified to electrophoretic homogeneneity from E. coli and its biochemical properties were studied. It differs from the previously studied endoglucanases A and B. In particular, endoglucanase C displays features common to endo- and exoglucanases, since it had a high activity on carboxymethylcellulose and on p-nitrophenyl-beta-D-cellobioside where only the agluconic bond was split. In addition, the enzyme was able to release cellobiose units from G3, G4 and G5 cellodextrins. Endoglucanase C was characterized by Western blot in a culture supernatant from C. thermocellum grown on cellulose, using an antiserum raised against the enzyme produced by E. coli.

Carboxymethylcellulose Sodium↗
PubMed1986

Hydration of cellobial by exo- and endo-type cellulases: evidence for catalytic flexibility of glycosylases.

New insight has been obtained into the catalytic capabilities of cellulase. Essentially homogeneous preparations of exo- (or Avicelase-) type and endo- (or CMCase-) type cellulases from Irpex lacteus and Aspergillus niger, respectively, were shown to hydrate the enolic bond of cellobial to form 2-deoxycellobiose. The A. niger enzyme also synthesized a small amount of a 2-deoxycellobiosyl-transfer product from cellobial. By use of digests conducted in deuterated buffer and 1H NMR spectra for product analysis, both cellulases were found to protonate (deuterate) the double bond of cellobial from below the si face of the D-glucal moiety, i.e., from a direction opposite that assumed for protonation of the beta-D-glycosidic linkages of cellulose and cellodextrins. The exo enzyme, which hydrolyzes the latter substrates primarily to cellobiose, rapidly catalyzed cellobial hydration to produce the beta-anomer of beta-D-glucopyranosyl(1----4)-2-deoxy-D-glucose-2(e)-d. The A. niger cellulase produced the same 2-deoxycellobiose-d from cellobial, though too slowly for its configuration to be determined. However, evidence was obtained for the formation of a beta-2-deoxycellobiosyl-d-D-glucose-transfer product by the enzyme. Thus, it is likely that all of the observed reactions with cellobial represent trans additions at the double bond. In any case, the anomeric configuration of products is created de novo. Separate mechanisms are described for the reaction of cellobial hydration and for the stereochemically different reaction of cellulose hydrolysis catalyzed by the present enzymes, assuming an arrangement of their catalytic groups analogous to that found in lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspergillus niger↗
PubMed1966

Cellulases of a marine mollusc, Dolabella sp.

1. A crude cellulase extract was prepared from the hepatopancreas of a marine mollusc, Dolabella sp., and partially purified by ammonium sulphate fractionation. 2. The optimum pH values of the partially purified preparation were 6.5 and 8.0 for Walseth cellulose and CM-cellulose respectively. It was most stable at pH6.0 and showed moderate thermostability. 3. The partially purified preparation was subjected to starch-zone electrophoresis, and incompletely resolved into several fractions that contained one or more cellulase components of different substrate specificity. 4. Some of these cellulase fractions showed practically no aryl beta-glucosidase activity and hydrolysed aryl beta-cellobioside with difficulty. From substrates such as higher cello-oligosaccharides, cellodextrin, CM-cellulose, Walseth cellulose and cotton fibre, they produced cellobiose as the major and cellotriose as the minor end products, both of which were resistant to further attack by cellulase. 5. From the slope of the curves of viscosity-reducing power for CM-cellulose, the cellulase components from Dolabella were presumed to be of a ;more-random' or a ;less-random' type in the mode of action. 6. In the hepatopancreas of this mollusc, beta-glucosidases were also present, which hydrolysed cellobiose as well as aryl beta-glucosides. The optimum pH values of these enzymes were about 5.5.

Animals↗
PubMed1973

Studies on beta-glucanases. Some properties of a bacterial endo-beta-(1 leads to 3)-glucanase system.

A commercial enzyme preparation, originally obtained from a Flavobacterium(Cytophaga), was fractionated by continuous electrophoresis, giving a protein fraction which hydrolysed laminarin, carboxymethylpachyman, barley beta-glucan, lichenin and cellodextrin in random fashion. This enzymic activity was not very stable. Ion-exchange chromatography and molecular-sieve chromatography on Bio-Gel P-60 showed that this activity was due to two specific beta-glucanases, an endo-beta-(1-->3)-glucanase and an endo-beta-(1-->4)-glucanase. The two enzymes occur in both high- and low-molecular-weight forms, the latter endo-beta-(1-->3)-glucanase having a molecular weight of about 16000.

Chromatography, Ion Exchange↗
PubMed1982

Characterization of a cellobiose dehydrogenase in the cellulolytic fungus Sporotrichum (Chrysosporium) thermophile.

An extracellular enzyme from culture filtrates of Sporotrichum (Chrysosporium) thermophile (A.T.C.C. 42 464) after growth on cellulose or cellobiose was shown to oxidize cellobiose to cellobionic acid in vitro. Lactose and cellodextrins were also efficiently oxidized, but the enzyme was not active against most mono- and di-saccharides. Several redox substances could act as electron acceptors, but molecular oxygen, tetrazolium salts and NAD(P) were not reduced. Activity was stimulated up to 2-fold in the presence of 0.05 M-Mg2+. The pH optimum of the enzymic reaction was acidic when the activity was tested with dichlorophenol-indophenol or Methylene Blue, but was neutral to alkaline for 3,5-di-t-butyl-1,2-benzoquinone or phenazine methosulphate as electron acceptors. As the enzyme was formed inductively in parallel with the endocellulase, its possible function in relation to cellulolysis is discussed.

Buffers↗
PubMed1983

Purification and properties of Cellvibrio gilvus cellobiose phosphorylase.

The cellobiose phosphorylase (EC 2.4.1.20) of Cellvibrio gilvus, which is an endocellular enzyme, has been purified 196-fold with a recovery of 11% and a specific activity of 27.4 mumol of glucose 1-phosphate formed/min per mg of protein. The purification procedure includes fractionation with protamine sulphate, and hydroxyapatite and DEAE-Sephadex A-50 chromatography. The enzyme appears homogeneous on polyacrylamide-gel electrophoresis, and a molecular weight of 280 000 was determined by molecular-sieve chromatography. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed a single band and mol.wt. 72 000, indicating that cellobiose phosphorylase consists of four subunits. The enzyme had a specificity for cellobiose, requiring Pi and Mg2+ for phosphorylation, but not for cellodextrin, gentibiose, laminaribiose, lactose, maltose, kojibiose and sucrose. The enzyme showed low thermostability, an optimum pH of 7.6 and a high stability in the presence of 2-mercaptoethanol or dithiothreitol. The Km values for cellobiose and Pi were 1.25 mM and 0.77 mM respectively. Nojirimycin acted as a powerful pure competitive inhibitor (with respect to cellobiose) of the enzyme (Ki = 45 microM). Addition of thiol-blocking agents to the enzyme caused 56% inhibition at 500 microM-N-ethylmaleimide and 100% at 20 microM-p-chloromercuribenzoate.

Catalysis↗
PubMed1990

Studies of the cellulolytic system of the filamentous fungus Trichoderma reesei QM 9414. Substrate specificity and transfer activity of endoglucanase I.

Endoglucanase I from the filamentous fungus Trichoderma reesei catalyses hydrolysis and glycosyl-transfer reactions of cello-oligosaccharides. Initial bond-cleaving frequencies determined with 1-3H-labelled cello-oligosaccharides proved to be substrate-concentration-dependent. Using chromophoric glycosides and analysing the reaction products by h.p.l.c., kinetic data are obtained and, as typical for an endo-type depolymerase, apparent hydrolytic parameters (kcat., kcat./Km) increase steadily as a function of the number of glucose residues. At high substrate concentrations, and for both free cellodextrins and their aromatic glycosides, complex patterns (transfer reactions) are, however, evident. In contrast with the corresponding lactosides and 1-thiocellobiosides, and in conflict with the expected specificity, aromatic 1-O-beta-cellobiosides are apparently hydrolysed at both scissile bonds, yielding the glucoside as one of the main reaction products. Its formation rate is clearly non-hyperbolically related to the substrate concentration and, since the rate of D-glucose formation is substantially lower, strong indications for dismutation reactions (self-transfer) are again obtained. Evidence for transfer reactions catalysed by endoglucanase I further results from experiments using different acceptor and donor substrates. A main transfer product accumulating in a digest containing a chromophoric 1-thioxyloside was isolated and its structure elucidated by proton n.m.r. spectrometry (500 MHz). The beta 1-4 configuration of the newly formed bond was proved.

Cellulase↗
PubMed1991

Purification and characterization of endoglucanase Ss from Clostridium thermocellum.

The extracellular cellulolytic enzymes of the thermophilic anaerobe Clostridium thermocellum occur as a protein complex or aggregate known as the cellulosome. By using a combination of ion-exchange, adsorption and hydrophobic-interaction chromatography, it was possible to isolate from extracellular broth a specific endoglucanase of interest without the use of denaturants. The endoglucanase was identified as the cellulosomal subunit Ss by the use of specific antibodies. The enzyme has an Mr of 83,000, an isoelectric point of 3.55, optimum pH of 6.6 and optimum temperature of 70 degrees C. It hydrolyses CM-cellulose and, at a higher rate, the cellodextrins, cellotetraose and cellopentaose, but does not hydrolyse a crystalline cellulose such as Avicel. Cellobiose and cellotriose are also immune to attack. It differs from endoglucanases previously isolated by others and a 76,000-Mr endoglucanase recently isolated in this laboratory.

Blotting, Western↗
PubMed1975

The site of cellulose synthesis. Hormone treatment alters the intracellular location of alkali-insoluble beta-1,4-glucan (cellulose) synthetase activities.

Membrane preparations from growing regions of 8-day old Pisum sativum epicotyls contain multiple beta-1,4-glucan (cellulose) synthetase activities (UDP- or GDP-glucose: beta-1,4-glucan-glucosyl transferase), and the levels of some of these are influenced by treatments with the growth hormone, indoleacetic acid (IAA). When membranes from control epicotyl segments (zero time) are fractionated by isopycnic sedimentation in sucrose density gradients, all of the synthetase activities are associated mainly with Golgi membrane (density 1.55 g/cm3). After decapitation and treatment of epicotyls with IAA, synthetases also appear in a smooth vesicle fraction (density 1.11 g/cm3) which is rich in endoplasmic reticulum (ER) marker enzyme. Major fractions of these synthetases are not recovered in association with plasma membrane or washed cell walls. When [14-C]sucrose is supplied in vivo to segments +/- IAA, radioactive cellulose is deposited only in the wall. Cellulose or cellodextrin precursors do not accumulate in those membranes in which synthetase activities are recovered in vitro. In experiments where tissue slices containing intact cells are supplied with [14C]sugar nucleotide in vitro, alkali-insoluble beta-1,4-glucan is synthesized (presumably outside the protoplast) at rates which greatly exceeded (20-30 times) those obtained using isolated membrane preparations. Progressive disruption of cell structure results in increasing losses of this high activity. These results are consistent with the interpretation that Golgi and ER-associated synthetases are not themselves loci for cellulose synthesis in vivo, but represent enzymes in transit to sites of action at the wall:protoplast omterface. There they operate only if integrity of cellular organization is maintained.

Cell Membrane↗
PubMed1975

Purification and properties of an exo-cellulase component of novel type from Trichoderma miride.

An enzyme extract from Cellulase-Onozuka, a commercial product of Trichoderma viride, was fractionated by Amberlite CG-50 column chromatography into three cellulase [EC 3.2.1.4] groups, peaks I to III. A noval enzyme, which has both beta-glucosidase [EC 3.2.1.21] and exo-carboxymethyl-cellulase (exo-CMCase) properties was obtained from peak III by extensive purification throuh consecutive column chromatography. The enzyme was homogeneous on ultracentrifugation, SDS-gel and cellulose acetate film electrophoreses and molecular sieve chromatography on Bio-Gel P-150. The molecular weight of this enzyme was estimated to be 53,000. The enzyme appeared to release cellobiose residues one by one from the nonreducing end of higher cellooligosaccharides and CM-cellulose (CMC), but to release glucosyl residues from reduced cellotriose and beta-cellobioside, resembling a beta-glucosidase in this respect. Furthermore, this exo-CMCase also attacked xylan exo-wise to produce xylobiose moleculaes one by one, but it scarcely attacked insoluble cellulose, except for a cellodextrin apparently rich in amorphous structure.

Animals↗
PubMed1992

Cloning and expression of multiple cellulase cDNAs from the anaerobic rumen fungus Neocallimastix patriciarum in Escherichia coli.

A cDNA expression library of the rumen fungus Neocallimastix patriciarum was made in Escherichia coli. Cellulolytic clones were identified by screening on a medium containing carboxymethylcellulose. Restriction mapping and Southern hybridization analysis of selected clones revealed three distinct cellulase cDNAs, designated celA, celB and celC. Studies on the substrate specificity showed that the enzyme encoded by celA had high activity towards amorphous and microcrystalline cellulose, while the celB and celC enzymes had relatively high activity on carboxymethylcellulose, with little activity on microcrystalline cellulose. Analysis of hydrolysis products from defined cellodextrins showed that the celB and celC enzymes hydrolysed beta-1,4-glucosidic linkages randomly, whereas the celA enzyme cleaved cellotetraose to cellobiose, and cellopentaose to cellobiose and cellotriose. Cellobiose was also the only product detectable from hydrolysis of microcrystalline cellulose by the celA enzyme. Based on substrate specificity and catalytic mode, celA appears to encode a cellobiohydrolase, while celB and celC encode endoglucanases. Northern blot hybridization analysis showed that expression of the three cellulase transcripts in N. patriciarum was induced by cellulose.

Animals↗