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At least 919 records · Page 51Linked to original sources
PubMed1979

Studies on cellulases of a phytopathogenic fungus, Pyricularia oryzae Cavara. III. Multiplicity of beta-glucosidase, and purification and properties of a second component.

To determine the relationship between the induction patterns of three components of beta-glucosidase of Pyricularia oryzae and carbon sources in the growth medium, various culture conditions were examined. Avicel, hydroxyethylcellulose and methyl-beta-D-glucoside as the carbon source induced both beta-glucosidase components, GB-1 and GB-2, whereas cellobiose and gentiobiose induced only one component, GB-1. Thus, these two components were induced independently and hence thought to be isozymes. The GB-2 was purified to homogeneity by ion exchange and gel filtration chromatographies from two different cultures on methyl-beta-D-glucoside and Avicel. The specific activity of GB-2 when salicin was used as substrate was approximately 5.9 mg glucose/min/mg protein. GB-2 was found to be an oligomeric glycoprotein, which consisted of two subunits with molecular weight of approximately 120,000, comprising a relatively large number of acidic amino acids and mannose, as is the case with GB-1. These two isozymes were clearly different in thermostability, GB-2 being more thermolabile than GB-1. However, the same carboxyl group (pKa 4.2--4.8) was found to be strongly implicated in the formation and dissociation of the enzyme-substrate complex for both of the enzymes, from the analysis of kinetic parameters as a function of pH.

Amino Acids↗
PubMed2001

Thermostabilization by replacement of specific residues with lysine in a Bacillus alkaline cellulase: building a structural model and implications of newly formed double intrahelical salt bridges.

An alkaline, mesophilic endo-1,4-beta-glucanase from alkaliphilic Bacillus sp. strain KSM-64 was significantly thermostabilized by replacement of both Asn179 and Asp194 with lysine by site-directed mutagenesis. Structural remodeling of the mutant enzyme newly generated by the double mutation suggested that Glu175-->Lys179 and Glu190-->Lys194 were the most plausible ion pairs, both of which involved side chains at the i and i + 4 positions on the alpha(4)-helix from Glu175 to Ser195. By molecular dynamics simulations, the N(zeta) hydrogens of Lys179 and Lys194 were found to coordinate with the carbonyl O(varepsilon1) and O(varepsilon2) of Glu175 and the carbonyl O(varepsilon1) of Glu190, respectively, with distances of around 2 A for all. These results confirm that the formation of these double intrahelical ion pairs (salt bridges) is responsible for the thermostabilization by the double mutation.

Alkalies↗
PubMedDate not supplied

Characterization and disruption of a gene in the maize pathogen Cochliobolus carbonum encoding a cellulase lacking a cellulose binding domain and hinge region.

A gene, CEL1, in the maize pathogen Cochliobolus carbonum was identified using the cbh1-3 gene of Phanerochaete chrysosporium as a heterologous probe. The predicted product of CEL1, Cel1, is 62% identical and 71% similar to the product of cbh1-3 and 54 to 62% identical to five cellobiohydrolases from other filamentous fungi. The location of the polyadenylation site 221 bp downstream of the stop codon and the location of a single intron of 55 bp were identified by comparison of the sequences of genomic and cDNA copies of CEL1. The transcriptional start site was determined by rapid amplification of cDNA ends (RACE) to be 39 bp upstream of the putative translational start site. CEL1 mRNA abundance is high when C. carbonum is grown on cellulose or maize cell walls but is undetectable when grown on 2% sucrose or cellulose plus sucrose. Cel1 has a predicted signal peptide of 18 amino acids and therefore a mature size of 46.4 kDa. Like the product of cbh1-1 of P. chrysosporium, but unlike most other endoglucanases and cellobiohydrolases (including the predicted product of cbh1-3), Cel1 does not have a putative cellulose binding domain or associated hinge region. The codon bias of CEL1 is stronger than the bias of cbh1-1 and comparable to that of cbh1-3 and that of the C. carbonum genes PGN1 and XYL1, (encoding endopolygalacturonase and endo-xylanase, respectively). A strain of C. carbonum specifically mutated at CEL1 was produced by transformation with a truncated copy of CEL1. Integration and disruption of CEL1 in the mutant was confirmed by DNA and RNA blotting. Pathogenicity of the CEL1 mutant was indistinguishable from the wild-type, indicating that CEL1 by itself is not a critical disease determinant. Culture filtrates of C. carbonum grown on cellulose or maize cell walls had several cellobiohydrolase, endoglucanase, and beta-glucosidase activities that were separable by chromatofocusing, hydrophobic interaction, or ion-exchange high-performance liquid chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2000

Streptomyces thermocoprophilus sp. nov., a cellulase-free endo-xylanase-producing streptomycete.

The taxonomic position of a thermophilic actinomycete strain isolated from poultry faeces was examined using a polyphasic approach. The isolate, designated B19T, was assigned to the genus Streptomyces on the basis of chemotaxonomic and morphological criteria. An almost complete 16S rRNA gene (rDNA) sequence obtained for the test strain was compared with those of representative streptomycetes, notably thermophilic streptomycetes. 16S rDNA sequence data not only supported the assignment of the strain to the genus Streptomyces but also showed that the isolate formed a distinct phyletic line within the evolutionary branch composed of Streptomyces thermodiastaticus and related species. The strain was distinguished from related validly described Streptomyces species by a number of phenotypic properties. It is, therefore, proposed that strain B19T be classified in the genus Streptomyces as Streptomyces thermocoprophilus sp. nov.

Animals↗
PubMed1989

The isolation and comparison of cellulase genes from two strains of Ruminococcus albus.

Endo-1,4-beta-glucanase genes have been cloned from two strains of Ruminococcus albus recently isolated in this laboratory. Although the strains were phenotypically similar, cross-hybridization studies between them showed significant genetic differences, with only 20% of the genome forming DNA heteroduplexes. Heteroduplexes displayed an average dissociation temperature 9 degrees C lower than that of the homoduplex. Consistent with this, restriction maps of the two endoglucanase genes showed no similarity, and hybridization work using the endoglucanase genes as probes revealed that neither gene was present in the genome of the other isolate of R. albus. Comparative enzyme characterization showed differences between the enzymes in their response to temperature, pH and substrate preference.

Animals↗
PubMed2006

Degradation of cellulose and hemicelluloses by the brown rot fungus Piptoporus betulinus--production of extracellular enzymes and characterization of the major cellulases.

Piptoporus betulinus is a common wood-rotting fungus parasitic for birch (Betula species). It is able to cause fast mass loss of birch wood or other lignocellulose substrates. When grown on wheat straw, P. betulinus caused 65% loss of dry mass within 98 days, and it produced endo-1,4-beta-glucanase (EG), endo-1,4-beta-xylanase, endo-1,4-beta-mannanase, 1,4-beta-glucosidase (BG), 1,4-beta-xylosidase, 1,4-beta-mannosidase and cellobiohydrolase activities. The fungus was not able to efficiently degrade crystalline cellulose. The major glycosyl hydrolases, endoglucanase EG1 and beta-glucosidase BG1, were purified. EG1 was a protein of 62 kDa with a pI of 2.6-2.8. It cleaved cellulose internally, produced cellobiose and glucose from cellulose and cellooligosaccharides, and also showed beta-xylosidase and endoxylanase activities. The K(m) for carboxymethylcellulose was 3.5 g l(-1), with the highest activity at pH 3.5 and 70 degrees C. BG1 was a protein of 36 kDa with a pI around 2.6. It was able to produce glucose from cellobiose and cellooligosaccharides, but also produced galactose, mannose and xylose from the respective oligosaccharides and showed some cellobiohydrolase activity. The K(m) for p-nitrophenyl-1,4-beta-glucoside was 1.8 mM, with the highest activity at pH 4 and 60 degrees C, and the enzyme was competitively inhibited by glucose (K(i)=5.8 mM). The fungus produced mainly beta-glucosidase and beta-mannosidase activity in its fruit bodies, while higher activities of endoglucanase, endoxylanase and beta-xylosidase were found in fungus-colonized wood.

Carboxymethylcellulose Sodium↗
PubMed1989

Conserved serine-rich sequences in xylanase and cellulase from Pseudomonas fluorescens subspecies cellulosa: internal signal sequence and unusual protein processing.

The complete nucleotide sequence of the xynA gene coding for a xylanase (XYLA) expressed by Pseudomonas fluorescens subspecies cellulosa, has been determined. The structural gene consists of an open reading frame of 1833 bp followed by a TAA stop codon. Confirmation of the nucleotide sequence was obtained by comparing the predicted amino acid sequence with that derived by N-terminal analysis of purified forms of the xylanase. The signal peptide present at the N terminus of mature XYLA closely resembles signal peptides of other secreted proteins. Truncated forms of the xylanase gene, in which the sequence encoding the N-terminal signal peptide had been deleted, still expressed coli. XYLA contains domains which are homologous to an endoglucanase expressed by the same organism. These structures include serine-rich sequences. Bal31 deletions of xynA revealed the extent to which these conserved sequences, in XYLA, were essential for xylanase activity. Downstream of the TAA stop codon is a G + C-rich region of dyad symmetry (delta G = 24 kcal) characteristic of E. coli Rho-independent transcription terminators.

Amino Acid Sequence↗
PubMed1993

Mutagenesis of cellulase EGZ for studying the general protein secretory pathway in Erwinia chrysanthemi.

Extracellular secretion of endoglucanase Z (EGZ) from Erwinia chrysanthemi is mediated by the so-called Out general secretion pathway and, presumably, involves recognition of EGZ-carried structural information by one or more of the Out proteins. Investigating the relationships between structure and secretability of EGZ was the purpose of the present work. EGZ is made of two independent domains, located at the N- and C-proximal sides, separated by a Ser/Thr-rich region, which are responsible for catalysis and cellulose-binding, respectively. The existence of a secretion region ('targeting signal') was investigated by studying the secretability of modified EGZ derivatives. These resulted from deletion or peptide insertion and were designed by using the domain organization cited above as a guide. Catalytic and/or cellulose-binding tests showed that all proteins exhibited at least a functional EGZ domain while immunoblot analyses confirmed that neither the insertions nor the deletions led to grossly misfolded proteins. In contrast, all of the proteins lost their secretability in E. chrysanthemi. This suggested that at least two secretion motifs existed, one lying within each functional domain. The role of the Ser/Thr-rich linker region was subsequently tested. Accordingly, two proteins containing a linker region whose length was increased by the addition of 8 and 18 additional residues and one protein lacking the linker region were studied. All three exhibited endoglucanase activity and cellulose-binding ability, confirming the independence of the domains within the context of EGZ/polysaccharide interaction. In contrast, none was secreted by E. chrysanthemi.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1996

Cloning of a cellulase gene from the rumen anaerobe Fibrobacter succinogenes SD35 and partial characterization of the gene product.

A gene encoding an enzyme which degrades cellulose (end-1) was isolated from a library of Fibrobacter succinogenes SD35 DNA fragments and expressed in pUC18. The product of end-1 showed significant activity against carboxymethylcellulose but relatively minor activity against lichenan, xylan and avicel. The nucleotide sequence indicated a product of 388 amino acids with a molecular mass of 50.2 kDa. This was in agreement with the molecular size estimated by gel electrophoresis. No significant DNA sequence similarity was identified with any published endoglucanase.

Amino Acid Sequence↗
PubMed1995

Cellulases and hemicellulases of the anaerobic fungus Piromyces constitute a multiprotein cellulose-binding complex and are encoded by multigene families.

More than 80% of the extracellular Avicelase, endoglucanase, xylanase and mannanase activities of the anaerobic fungus Piromyces were associated with a cellulose-binding complex. The complex was composed of at least 10 polypeptides ranging in size from 190 kDa to 50 kDa, and contained numerous endoglucanases, xylanases and mannanases. Multiple genes encoding each of these activities were isolated from an expressing cDNA library.

Anaerobiosis↗
PubMed1998

Two genes of the anaerobic fungus Orpinomyces sp. strain PC-2 encoding cellulases with endoglucanase activities may have arisen by gene duplication.

A cDNA designated celE cloned from Orpinomyces PC-2 consisted of an open reading frame encoding a polypeptide (CelE) of 477 amino acids. CelE was highly homologous to CelBs of Orpinomyces (72.3% identity) and neocallimastix (67.9% identity) and like them it had a non-catalytic repeated peptide domain (NCRPD) at the C-terminal end. The catalytic domain of CelE was homologous to glycosyl hydrolases of Family 5, found in several anaerobic bacteria. The gene of celE was devoid of introns. The recombinant proteins CelE and CelB of Orpinomyces PC-2 randomly hydrolyzed carboxymethylcellulose and cello-oligosaccharides in the pattern of endoglucanases. The results indicated that a gene of bacterial origin was duplicated to form celE and celB of Orpinomyces PC-2.

Amino Acid Sequence↗
PubMed2001

Identification of thermostabilizing residues in a Bacillus alkaline cellulase by construction of chimeras from mesophilic and thermostable enzymes and site-directed mutagenesis.

An alkaliphilic Bacillus sp. strain, KSM-64, produces a mesophilic alkaline endo-1,4-beta-glucanase that is suitable for use in detergents. The deduced amino acid sequence of the enzyme showed very high homology to that of a thermostable alkaline enzyme from alkaliphilic Bacillus sp. strain KSM-S237. Analysis of chimeric enzymes produced from the genes encoding the mesophilic and thermostable enzymes suggested that the lysine residues at positions 137, 179, and 194 are responsible for their thermal stabilization. Replacing the corresponding Glu137, Asn179, and/or Asp194 with lysine by site-directed mutagenesis made the mesophilic enzyme more thermostable. Analyses of the hydrophilicity of deduced amino acid sequences and isoelectric focusing of the modified enzymes suggested that these three specific lysine residues and their replacements are all located on the surface of the enzyme molecule. This fact further suggested that specific ionic interaction is involved in the thermal stabilization of the enzyme.

Alkalies↗