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At least 595 records · Page 33Linked to original sources
PubMed1986

Homology between cellulase genes of Trichoderma reesei: complete nucleotide sequence of the endoglucanase I gene.

The filamentous fungus Trichoderma reesei produces several endoglucanases (EG) and cellobiohydrolases (CBH) which are involved in cellulose hydrolysis in a complex synergistic manner. We have cloned and sequenced the gene and the full-length cDNA coding for the major endoglucanase EG-I, and compared this to the cbh1 gene sequence to clarify the relationship between the EG and CBH classes of cellulases. The deduced 437-amino acids (aa) long EG-I protein with a 22-aa long signal peptide is 45% identical in aa sequence with CBH-I. The best conserved region is found at the C terminus and shows about 70% homology. The data suggest that the two enzymes have arisen from a common ancestor by gene duplication. Despite this, the intron positions have not been conserved in these genes which both contain two short introns. The deduced EG-I sequence contains six putative N-glycosylation sites, and a putative O-glycosylated region is found near the C terminus, closely resembling a similar region at the C terminus of CBH-I. Comparison of the aa sequences suggests that the evolutionary divergence of EG-I from CBH-I has involved four separate 10-20 aa "deletions" from the ancestral protein.

Amino Acid Sequence↗
PubMed1988

Cloning and expression of two cellulase genes of Clostridium cellulolyticum in Escherichia coli.

Two cellulase genes isolated from Clostridium cellulolyticum strain ATCC3519 were cloned in Escherichia coli using plasmid pACYC184. Plasmids pB52 and pB43 were isolated from the transformants producing carboxymethylcellulase (CMCase) and the two cloned CMCase-coding genes were found to be included in two EcoRI fragments of 5.7 kb and 2.6 kb, respectively. These two genes showed no homology. The CMCase-coding genes were found to be contained in a 1.8-kb KpnI-HindIII fragment and a 2.05-kb HindIII-PvuII fragment of the DNA donor strain. Expression of these genes in E. coli was found not to depend on their orientation in the cloning vector. Hybridization experiments between these two fragments and Clostridium thermocellum NCIB10682 DNA fragments carrying genes celA, celB, celC and celD were carried out and some homologies were detected.

Cellulase↗
PubMed1991

Sequence analysis of the cellulase-encoding celY gene of Erwinia chrysanthemi: a possible case of interspecies gene transfer.

The Erwinia chrysanthemi (strain 3937) celY gene encoding the minor endoglucanase (EGY) was sequenced. The analysis of the upstream region allowed us to identify an in vivo active promoter recognized by the NtrA (sigma 54) holoenzyme. No similarity was found between the predicted amino acid (aa) sequences of EGY and either the Er. chrysanthemi major endoglucanase, EGZ, or the Er. carotovora CelS endoglucanase. In contrast, a very high level of identity, both at the nucleotide and the predicted aa levels, was found between celY and an EG-encoding gene from Cellulomonas uda, a Gram + bacterium taxonomically distant from Er. chrysanthemi. By comparing the molar G + C% of the cellulase-encoding genes and that of Er. chrysanthemi and C. uda chromosomal DNAs, we speculate that celY was transferred from Er. chrysanthemi to C. uda.

Amino Acid Sequence↗
PubMed1993

Secretion of a prokaryotic cellulase in bacterial and mammalian cells.

The catalytic domain of mature Clostridium thermocellum endoglucanase E (EGE') and derivatives of the enzyme fused to prokaryote and eukaryote signal peptides (SP), were produced in Chinese hamster ovary (CHO) cells and Escherichia coli. All three forms of the endoglucanase were secreted into the periplasm of Escherichia coli, but only derivatives of the enzyme containing an N-terminal SP were exported from CHO cells. Extracellular EGE', purified from E. coli and CHO cultures, displayed similar properties suggesting that glycosylation of the enzyme in the eukaryote did not significantly alter the protein's properties. Data presented in this report indicate that mature EGE' contains secretion signals which are recognised only by the E. coli protein export apparatus, suggesting that there are differences in the recognition of certain secretion signals in eukaryotes and prokaryotes. As mature EGE' does not contain secretion signals recognised by the mammalian cell, membrane translocation of the bacterial cellulase in a higher eukaryote is directed by an N-terminal prokaryotic SP.

Animals↗
PubMedDate not supplied

The effects of tunicamycin on secretion, adhesion and activities of the cellulase complex of Clostridium cellulolyticum, ATCC 35319.

The effects of tunicamycin, an inhibitor of N-asparagine-linked glycosylation, on the secretion, adhesion and activities of the cellulase complex produced by Clostridium cellulolyticum have been studied. Tunicamycin at 0.1 micrograms/ml slightly inhibited growth on cellobiose. Endoglucanase, p-nitrophenylcellobiosidase and avicelase activities of the "Avicel"-adsorbed fraction from a culture grown with this drug were decreased 4.4-, 1.4- and 12.2-fold, respectively. During growth on cellulose, tunicamycin considerably inhibited growth and adhesion of cells on their substrate (only 28% of the cells were bound to cellulose). SDS-PAGE mobilities of some proteins excreted during growth with the drug were different from those of proteins from control cultures; the native Avicel-adsorbed fraction (PH2O) consisted of three major components of molecular weights about 135, 90 and 68 kDa, whereas in the presence of tunicamycin (0.1 micrograms/ml), the Avicel-adsorbed fraction (PH2OT) contained only a major band of 105 kDa, and the proteins of 135 and 68 kDa appeared weakly. By using the "Dig Glycan Detection" kit, some proteins appeared to be glycosylated, such as the 135-, 95-, 47- and 40-kDa proteins. Moreover, the affinity for Avicel and the avicelase activity decreased dramatically for the Avicel-adsorbed fraction from a culture grown with the drug. The remaining avicelase activity of the PH2O fraction in the presence of specific P135 antiserum was 50% of the initial activity, whereas CMCase and pNPCbase were not affected. The glycosylated protein of 135 kDa played a prominent role in the adhesion and avicelase activity of C. cellulolyticum. Moreover, the endoglucanase activity in a culture broth from tunicamycin-grown cells was more thermolabile and protease-sensitive than that from control cultures.

Anti-Bacterial Agents↗
PubMed2005

N-glycosylation is necessary for enzymatic activity of a beetle (Apriona germari) cellulase.

We previously reported that the beta-1,4-endoglucanase (EGase) belonging to glycoside hydrolase family 45 cloned from the mulberry longicorn beetle, Apriona germari (Ag-EGase I), is composed of 237 amino acid residues and has a potential N-glycosylation site at 97-100 amino acid residues (NSTF). We here describe the N-glycosylation and its role for enzymatic activity of the Ag-EGase I. The N-glycosylation of Ag-EGase I was revealed by the treatment of tunicamycin to the recombinant virus-infected insect Sf9 cells and by endoglycosidase F to the purified recombinant Ag-EGase I, demonstrating that the carbohydrate moieties are not necessary for secretion but essential for Ag-EGase I enzyme activity. To further elucidate the functional role of the N-glycosylation in Ag-EGase I, we have assayed the cellulase enzyme activity in Thr99Gln mutant. Lack of N-glycosylation in Ag-EGase I showed no substantial enzyme activity. This result demonstrates that N-glycosylation at site 97-100 amino acid residues (NSTF) is essential for enzyme activity.

Animals↗
PubMed2006

Production of cellulase-free endoxylanase from novel alkalophilic thermotolerent Bacillus pumilus by solid-state fermentation and its application in wastepaper recycling.

The present study aimed at optimization of culture condition for the enhanced production of extra cellular thermostable cellulase-free xylanase from Bacillus pumilus by solid-state fermentation. Batch studies were carried out to evaluate various agro-industrial residues such as rice bran, rice husk, rice straw, sawdust, coconut pith, sugarcane bagasse and wheat bran for enzyme production by the bacterial culture. The endoxylanase production was highest on wheat bran media (5582 U/gds), which was enhanced 3.8-fold (21,431 U/gds) by optimization of cultivation conditions. The enzymatic extracts was used in mixed wastepaper recycling, which resulted in a considerable improvement of the paper strength with high drainage and easy drying up. The results of enzyme application with recycled paper clearly indicated that the effective use of enzymes in fiber separation could reduce the cost of carton paper production.

Bacillus↗
PubMed2006

Strain improvement of Penicillium janthinellum NCIM 1171 for increased cellulase production.

The strain of Penicillium janthinellum NCIM 1171 was subjected to mutation involving treatment of Ethyl Methyl Sulfonate (EMS) for 24h followed by UV-irradiation for 3min. Successive mutants showed enhanced cellulase production (EMS-UV-8), clearance zone on Avicel containing plate (SM2) and rapid growth on Walseth cellulose agar plates containing 0.2% 2-deoxy-d-glucose (SM3). These mutants were transferred to Walseth cellulose plates containing higher concentration (1.5%) of 2-deoxy-d-glucose (SM4) in which only five mutants showed clearance zone on SM4. All these mutants showed approximately two-fold increase in activity of both FPase and CMCase in shake flask culture when grown on basal medium containing CP-123 (1%) and wheat bran (2.5%). The enzyme preparations from these mutants were used to hydrolyze Avicel. Higher hydrolysis yields of Avicel were obtained with enzyme preparations of EU1. This is the first report on the isolation and selection of mutants based on hydrolysis of Avicel, which is the most crystalline substrate.

Cellulase↗
PubMed2004

Quantitative estimate of the effect of cellulase components during degradation of cotton fibers.

A comprehensive mechanistic kinetic model for enzymatic degradation of cotton fibers has been established based on a complete factorial experiment in combination with multivariate stepwise regression analysis. The analysis of the statistical parameter value in the model suggests that the enzymatic degradation of cotton fiber is a progressive and heterogeneous process that includes, at least, two courses that occur sequentially and then progress in parallel. Cellulose fibers were first depolymerized or solubilized by the synergism between cellobiohydrolase I (CBHI) and endoglucanase I (EGI), and then the oligomers obtained were randomly hydrolyzed into glucose by EGI and beta-glucosidase. The proposed model can be applied to the quantitative estimation of the effects of three cellulase components, CBHI, EGI, and beta-glucosidase separately, or in combination during the entire process of cellulose degradation. The validity of the proposed model has been verified by a filter paper activity assay. Its other applicability was also discussed.

Cellulase↗
PubMed2006

CP/MAS 13C NMR analysis of cellulase treated bleached softwood kraft pulp.

Fully bleached softwood kraft pulps were hydrolyzed with cellulase (1,4-(1,3:1,4)-beta-D-glucan 4-glucano-hydrolase, EC 3.2.1.4) from Trichoderma reesei. Supra-molecular structural features of cellulose during enzymatic hydrolysis were examined by using CP/MAS 13C NMR spectra in combination with line-fitting analysis. Different types of cellulose allomorphs (cellulose I(alpha), cellulose I(beta), para-crystalline) and amorphous regions were hydrolyzed to a different extent by the enzyme used. Also observed was a rapid initial phase for hydrolysis of regions followed by a slow hydrolysis phase. Cellulose I(alpha), para-crystalline, and non-crystalline regions of cellulose are more susceptible to enzymatic hydrolysis than cellulose I(beta) during the initial phase. After the initial phase, all the regions are then similarly susceptible to enzymatic hydrolysis.

Carbon Isotopes↗
PubMed2004

A family of glycosyl hydrolase family 45 cellulases from the pine wood nematode Bursaphelenchus xylophilus.

We have characterized a family of GHF45 cellulases from the pine wood nematode Bursaphelenchus xylophilus. The absence of such genes from other nematodes and their similarity to fungal genes suggests that they may have been acquired by horizontal gene transfer (HGT) from fungi. The cell wall degrading enzymes of other plant parasitic nematodes may have been acquired by HGT from bacteria. B. xylophilus is not directly related to other plant parasites and our data therefore suggest that horizontal transfer of cell wall degrading enzymes has played a key role in evolution of plant parasitism by nematodes on more than one occasion.

Amino Acid Sequence↗
PubMed2005

Model cellulose films exposed to H. insolens glucoside hydrolase family 45 endo-cellulase--the effect of the carbohydrate-binding module.

The effects of enzyme structure and activity on the degradation of model cellulose substrates were investigated by ellipsometry for the cellulase Humicola insolens GH45. The inactive variant D10N was found to adsorb at the cellulose surface but also to be incorporated into the cellulose films to an extent that depended on pH. For the native protein, the initial adsorption monitored for the inactive variant D10N was followed by enzyme-mediated degradation of the cellulose films. Again, a dependence on pH was found, such that higher pH resulted in slower enzymatic degradation. Removing the carbohydrate-binding module eliminated this pH dependence but also resulted in a decreased adsorption to the cellulose surface, and in a decreased net catalytic effect.

Ascomycota↗
PubMed1986

Direct 1H n.m.r. determination of the stereochemical course of hydrolyses catalysed by glucanase components of the cellulase complex.

The stereochemical courses of the hydrolyses catalysed by three glycosidases have been determined directly by 1H nmr. The anomeric configuration of the initially formed product was ascertained in each case by observation of the chemical shift and coupling constant of the anomeric proton at the new hemiacetal centre. Two of the enzymes investigated, an endo-glucanase and an exo-glucanase are components of the cellulase complex of Cellulomonas fimi. The third enzyme is the beta-glucosidase from almond emulsin. Two of these enzymes, the exo-glucanase and the almond beta-glucosidase catalysed hydrolysis with retention of anomeric configuration, in agreement with previous observations on the almond enzyme. The endo-glucanase catalysed hydrolysis with inversion of configuration, this result being confirmed by optical rotation measurements. This 1H nmr approach has several advantages over other techniques in that it is applicable to a wide variety of glycosidases and substrates and it is non-destructive, allowing recovery of the enzyme.

Cellobiose↗
PubMed2002

Effects of cellulase on the modification of cellulose.

Multicomponent cellulases, purified endoglucanases and cellobiohydrolases were assayed and shown to modify pure natural cellulose (softwood pulp). Changes in structure and properties of the cellulose caused by enzymatic treatment depend on the composition, the type of enzyme, and the treatment conditions. The reactivity of cellulose for some dissolving and derivatization processes may be improved by enzymatic hydrolysis. Endoglucanases decreased the average degrees of polymerization (DP) and improved the alkaline solubility of cellulose most efficiently. The variation in the supramolecular structure estimated from the infrared spectra of the cellulose samples was found to be correlated with the reactivity and might represent wide variations in conformation caused by the breakdown of the hydrogen bonds.

Carboxymethylcellulose Sodium↗
PubMed1999

Transglycosylation of cellobiose by partially purified Trichoderma viride cellulase.

A commercial cellulase from Trichoderma viride was fractionated into three fractions, F1, F2, and F3, in order to investigate transglycosylation activities. Among these fractions, F3, which demonstrated highly hydrolytic activity toward p-nitrophenyl beta-D-glucopyranoside and Avicel, most effectively catalyzed the transglycosylation of cellobiose and converted cellobiose into beta-Glc-(1-->6)-beta-glc-(1-->4)-Glc and beta-Glc-(1-->6)-beta-Glc-(1-->6)-beta-Glc(1-->4)-Glc. The F3 fraction contained the enzyme to catalyze beta-glucosyl transfer toward only the C-6 position of the sugar acceptor, and thus it is expected to be of use for syntheses of functional oligosaccharides.

Carbohydrate Sequence↗
PubMed1997

Interaction between cellohexaose and cellulose binding domains from Trichoderma reesei cellulases.

Most Trichoderma reesei cellulases consist of a catalytic and a cellulose binding domain (CBD) joined by a linker. We have used cellohexaose as a model compound for the glucose chain to investigate the interaction between the soluble enzyme and cellulose. The binding of cellohexaose to family I CBDs was studied by NMR spectroscopy. CBDs cause line broadening effects and decreasing T2 relaxation times for certain cellohexaose resonances, whereas there are no effects in the presence of a mutant which binds weakly to cellulose. Yet it remains uncertain how well the soluble cellooligosaccharide mimics the binding of CBD to the cellulose.

Binding Sites↗
PubMed1999

Multiple endo-1,4-beta-D-glucanase (cellulase) genes in Arabidopsis.

The plant cell wall is modified in coordination with almost all plant developmental processes. Modifications in the cell wall are thought to be mediated by cell wall hydrolases, including those encoded by a large family of genes specifying endo-1,4-beta-D-glucanases (EC 3.2.1.4), which participate in the breakdown of beta-1,4 glucosidic linkages. The enzymes expected to modify cellulose, commonly referred to as cellulases, are encoded by members of this gene family. In Arabidopsis the endo-1,4-beta-D-glucanase (EGase) gene family is extensive (more than 12 members) and encompasses structurally different classes of genes encoding proteins with contrasting enzyme functions. Within the family there are enzymes located at the plasma membrane that are presumed to act at the innermost layers of the cell wall, and enzymes that are secreted and are presumed to act at any stratum within the cell wall, including the outermost layer. Both structural gene groups are members of the glycosyl hydrolase gene Family 9. Evidence suggests that EGases anchored in the plasma membrane play a role in cell wall biosynthetic processes, presumably by editing cellulose synthesis or during the assembly of the cellulose-hemicellulose network. Those EGases that are extracellular play specific roles in cell wall catabolic processes and their activity ranges from partial and localized to massive and catastrophic. This range in activity is linked to processes such as cell growth and cell death, respectively. For all Arabidopsis EGases nothing is known about their true in vivo substrate, mode of action, or to what extent they can act on cellulose or other beta-1,4 glucans. The study of the EGase gene family is in its infancy, and because of the possible agronomic implications this group of genes deserves continued attention.

Amino Acid Sequence↗
PubMed1999

Metazoan cellulase genes from termites: intron/exon structures and sites of expression.

Endogenous endo-beta-1,4-glucanase (EGase, EC 3.2.1.4) cDNAs were cloned from representatives of the termite families Termitidae and Rhinotermitidae. These EGases are all composed of 448 amino acids and belong to glycosyl hydrolase family 9 (GHF9), sharing high levels of identity (40-52%) with selected bacterial, mycetozoan and plant EGases. Like most plant EGases, they consist of a single catalytic domain, lacking the ancillary domains found in most microbial cellulases. Using a PCR-based strategy, the entire sequence of the coding region of NtEG, a gene putatively encoding an EGase from Nasutitermes takasagoensis (Termitidae), was determined. NtEG consists of 10 exons interrupted by 9 introns and contains typical eukaryotic promoter elements. Genomic fragments of EGase genes from Reticulitermes speratus (Rhinotermitidae) were also sequenced. In situ hybridization of N. takasagoensis guts with an antisense NtEG RNA probe demonstrated that expression occurs in the midgut, which contrasts to EGase expression being detected only in the salivary glands of R. speratus. NtEG, when expressed in Escherichia coli, was shown to have in vitro activity against carboxymethylcellulose.

Animals↗