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Query: UNIPROT:P06889 (Mol)
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Three cDNA clones (pCel 10, pCel 20 and pCel 30), each encoding different endo-beta-1,4-glucanases in peach, were obtained by RT-PCR and their expression investigated by northern analysis during leaf and fruit abscission and during fruit development. This analysis allowed the detection of only the pCel 10-related mRNA. A 2.2 kb transcript accumulated in ethylene activated abscission zones of leaves and fruits, and ppEG1 (Prunus persica endoglucanase 1) the gene coding for pCel 10, was isolated and characterized. A cDNA (termed pCel 1), containing the entire open reading frame of ppEGC1, was obtained and its sequence used to define the structure of the gene and the exon/intron boundaries. ppEG1 consists of 7 exons and encodes a 497 amino acid polypeptide including a putative signal peptide at the N-terminus. The similarity of this peach endo-beta-1,4-glucanase (EGase, EC 3.2.1.4) is high (76.3%) with the ripening avocado and low (47.3%) with the bean abscission EGase. A 1639 bp region at the 5' of the transcription start site shows regulatory functions in transgenic tobacco plants, as judged by its ability to drive GUS expression in cell separation-related events.
Plant Mol Biol 1997 Jul
PMID:Characterization of ppEG1, a member of a multigene family which encodes endo-beta-1,4-glucanase in peach. 927 69

The isolation of an elongation-specific endo-1,4-beta-glucanase-cel1 from Arabidopsis thaliana was made possible by the fact that considerable homology exists between different endo-1,4-beta-glucanase (EGase) genes from different plants. Degenerate primers were synthesized based on two conserved regions from the avocado and tomato cellulase amino acid sequences. The A.thaliana cel1 cDNA gene was found to encode a 54kDa protein; sequence comparison with the avocado EGase revealed 56% identity. Northern blot analysis of cel1 suggested its developmental regulation. RNA transcripts were undetectable in fully expanded leaves as well as at the basal internode of flowering stems. However, a strong transcript signal was detected in the elongating zone of flowering stems of normal plants. The RNA transcript level of cel1 in the elongating zone of dwarf flowering stems was significantly lower than in the corresponding zone in normal plants. This suggests cel1's involvement in cell elongation in A. thaliana. Transgenic tobacco plants transformed with the putative cel1 promoter region fused to the gus reporter gene, showed a significant GUS staining both in shoot and root elongating zones. These results further substantiate the link between cel1 expression and plant cell elongation.
Plant Mol Biol 1997 Aug
PMID:Cloning and characterization of elongation specific endo-1,4-beta-glucanase (cel1) from Arabidopsis thaliana. 929 Jun 36

The marine rotifer, Brachionus plicatilis, is able to digest Chlorella efficiently, suggesting that the rotifer contains a powerful cellulolytic enzyme system. A multi-component cellulolytic complex, including endoglucanase (CM-cellulase), cellobiohydrolase and beta-glucosidase, was found in Brachionus plicatilis. Endoglucanase (endo-beta-1,4 glucanase) was purified to homogeneity from rotifer homogenates using a sequential chromatographic method. The purified enzyme exhibits a strong hydrolytic activity with carboxymethyl(CM)-cellulose. The optimum temperature and pH for the endoglucanase activity were 37 degrees C and 7.0, respectively. 80% of the CM-cellulase activity was retained in salt mixture that ranged from 150 to 500 mM NaCl equivalent. The purified protein was isolated with a molecular weight of approximately 62 kDa estimated by SDS-polyacrylamide gel electrophoresis.
Biochem Mol Biol Int 1997 Oct
PMID:Purification and characterization of an endoglucanase from the marine rotifer, Brachionus plicatilis. 935 Mar 31

Detailed information has been obtained, by means of protein X-ray crystallography, on how a cellulose chain is bound in the cellulose-binding tunnel of cellobiohydrolase I (CBHI), the major cellulase in the hydrolysis of native, crystalline cellulose by the fungus Trichoderma reesei. Three high-resolution crystal structures of different catalytically deficient mutants of CBHI in complex with cellotetraose, cellopentaose and cellohexaose have been refined at 1.9, 1.7 and 1.9 A resolution, respectively. The observed binding of cellooligomers in the tunnel allowed unambiguous identification of ten well-defined subsites for glucosyl units that span a length of approximately 50 A. All bound oligomers have the same directionality and orientation, and the positions of the glucosyl units in each binding site agree remarkably well between the different complexes. The binding mode observed here corresponds to that expected during productive binding of a cellulose chain. The structures support the hypothesis that hydrolysis by CBHI proceeds from the reducing towards the non-reducing end of a cellulose chain, and they provide a structural explanation for the observed distribution of initial hydrolysis products.
J Mol Biol 1998 Jan 16
PMID:High-resolution crystal structures reveal how a cellulose chain is bound in the 50 A long tunnel of cellobiohydrolase I from Trichoderma reesei. 946 11

Compost is the preferred substrate for growth of the edible fungus Agaricus bisporus. Utilization of compost requires the production of enzymes involved in degradation of lignocellulolytic components. For molecular characterization of these processes we are isolating the encoding genes. By applying heterologous screening techniques, we have cloned such a gene, which is specifically induced on compost encoding an endo-1,4-beta-xylanase (xlnA) belonging to glycosyl hydrolase family 10. The gene encodes a pre-protein of 333 amino acid residues with a predicted molecular mass of 34,946 for the mature protein. The open reading frame is interrupted by ten introns of which introns 5 and 6 are separated by an exon of only two base-pairs. High expression of the xlnA gene was observed in vegetative mycelium grown on sterilized compost while xlnA messengers were not detected in fruit bodies. Addition of glucose or xylose to compost repressed xlnA expression. When glucose-grown colonies were transferred to a medium containing cellulose, xylan or xylose as sole carbon source, the organism responded by expressing xlnA at a high level for a short period. Transfer from glucose to compost yielded a much stronger and constant xlnA induction. A similar pattern of expression was found for the cel3 gene encoding a cellulase, suggesting that these genes are induced by compost-specific factors rather than by the substrates they act upon. Antiserum raised against XLNA protein, which was heterologously expressed in Escherichia coli, detected, when the fungus was grown on compost, an extracellular protein of 33 kDa with endo-xylanase activity.
J Mol Biol 1998 Mar 27
PMID:An endo-1,4-beta-xylanase-encoding gene from Agaricus bisporus is regulated by compost-specific factors. 951 54

The phytopathogenic Erwinia carotovora subsp. carotovora LY34 secretes multiple isozymes of the plant cell wall-disintegrating enzyme, endoglucanases. Genomic DNA from Ecc LY34 was digested with Sau3AI and ligated into the BamHI site of pBluescript II SK+. One of the E. coli clones containing a Sau3AI fragment of Ecc genomic DNA hydrolyzed carboxymethyl cellulose and was shown to contain the 2.2 kb BamHI restriction fragment, which was subcloned to generate pLYCA100 named as celA. The structural organization of a celA gene encoding 387 amino acids consists of an open reading frame (ORF) of 1161 bp starting with an ATG start codon and followed by a TAA stop codon. CelA protein contained a typical catalytic domain, interdomain, cellulose binding domain, and prokaryotic signal peptide of 32 amino acids. Since the deduced amino acid sequences of CelA protein was very similar to those of CelV of Erwinia carotovora subsp. carotovora SCC3193 enzyme and to those of CelN of Erwinia atroceptica enzyme, it belongs to the cellulase family 5. The apparent molecular mass of CelA protein was calculated to be 39 kDa by carboxymethylcellulose-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (CMC-SDS-PAGE). Activity staining of carboxymethyl cellulase (CMCase) in sodium dodecyl sulfate polyacrylamide gel containing 0.1% CMC revealed that the cloned isozyme comigrated with a corresponding isozyme produced by Ecc LY34. The CelA had a calculated pI of 5.42. The optimum pH was 7 and the optimum temperature was about 45 degrees C.
Mol Cells 1998 Feb 28
PMID:Cloning and sequencing of the celA gene encoding CMCase of Erwinia carotovora subsp. carotovora LY34. 957 28

A decade ago, Pugsley and colleagues reported the existence of a large region of Klebsiella DNA, distinct from the Klebsiella gene encoding pullulanase, which was necessary for secretion of this enzyme to the cell surface in Escherichia coli (d'Enfert et al., 1987a,b). The pul genes it contained proved to be the tip of an iceberg. The sequences reported before 1992 (d'Enfert et al., 1987a,b; d'Enfert & Pugsley, 1989; Pugsley & Reyss, 1990; Reyss & Pugsley, 1990) included only one gene (pulD) that matched any sequence in the data base; a 220 amino acid residue segment of PulD was 32% identical with a portion of the filamentous phage-encoded protein, pIV. But by the time the sequence of the 18.8 kb DNA fragment that contained the pul genes had been completed (Possot et al., 1992), reports of sets of homologous genes in several species of Gram-negative plant and animal pathogens had appeared. For the most part, these gene clusters were cloned by their ability to complement mutants that produced, but failed to secrete, proteins normally found in the extracellular milieu; when tested, the mutants showed reduced pathogenicity or were totally avirulent. The secreted proteins included hydrolytic enzymes such as cellulase and pectinase from plant pathogens, and proteases and toxins from animal pathogens. The multi-gene family necessary for secretion of these enzymes is now known as the type II system or the main terminal branch (MTB) of the general secretion pathway (GSP). As summarized by Pugsley et al. (1997), the current tally includes type II systems from Klebsiella oxytoca (pul), Erwinia chrysanthemi and carotovora (out), Xanthomonas campestris (xps), Pseudomonas aeruginosa (xcp), Aeromonas hydrophila (exe), and Vibrio cholerae (eps). A second type II system (sps) necessary for deposition of the S-layer on the cell surface in A. hydrophila is more similar to the X. campestris than A. hydrophila genes (Thomas & Trust, 1995). The biggest surprise has been the discovery of a complete set of type II secretion genes in E. coli K12. The E. coli genes are not expressed under normal growth conditions, and a search is underway to find inducing conditions and secretion substrates (Francetic & Pugsley, 1996). Impressive progress has already been made in defining components of the pathway. What remains to be understood in mechanistic detail is how this protein secretion system functions.
J Mol Biol 1998 Jun 12
PMID:Macromolecular assembly and secretion across the bacterial cell envelope: type II protein secretion systems. 964 73

We have identified a gene important for the regulation of exoenzyme virulence factor synthesis in the plant pathogen Erwinia carotovora ssp. carotovora (Ecc) and virulence and motility in Erwinia carotovora ssp. atroseptica (Eca). This gene, hexA (hyperproduction of exoenzymes), is a close relative of the Erwinia chrysanthemi (Echr) gene pecT and encodes a member of the LysR family of transcriptional regulators. hexA mutants in both Ecc and Eca produce abnormally high levels of the exoenzyme virulence factors pectate lyase, cellulase and protease. In addition, Eca hexA mutants show increased expression of the fliA and fliC genes and hypermotility. Consistent with a role as a global regulator, expression of hexA from even a low-copy plasmid can suppress exoenzyme production in Ecc and Eca and motility in Eca. Production of the quorum-sensing pheromone OHHL in Ecc hexA is higher throughout the growth curve compared with the wild-type strain. Overexpression of Ecc hexA also caused widespread effects in several strains of the opportunistic human pathogen, Serratia. Low-copy hexA expression resulted in repression of exoenzyme, pigment and antibiotic production and repression of the spreading phenotype. Finally, mutations in hexA were shown to increase Ecc or Eca virulence in planta.
Mol Microbiol 1998 May
PMID:The hexA gene of Erwinia carotovora encodes a LysR homologue and regulates motility and the expression of multiple virulence determinants. 964 39

A gene encoding cellulase-free xylanase was cloned from the thermophilic bacterium Bacillus sp. KK-1 into Escherichia coli and the gene product was purified from the recombinant E. coli. This xylanase gene, designated xylY, was composed of 1,302 base pairs and encoded a polypeptide of 434 amino acids. No similarity was found between the nucleotide sequence of the xylY gene and those reported for other xylanase genes. The deduced amino acid sequence was homologous to those of cellulases belonging to the beta-glycanase family D. The purified enzyme exhibited maximum activity at 55 degrees C but also lost 70% of this activity even after incubation for 30 min at 55 degrees C. Bacillus sp. KK-1 may have acquired the xylY gene by an interspecies gene transfer during adaptation to mesophilic environment.
Biochem Mol Biol Int 1998 Jun
PMID:Molecular cloning of a Bacillus sp. KK-1 xylanase gene and characterization of the gene product. 967 55

A bacteria identified as Bacillus brevis has been isolated from the soil. It has been found to secrete cellulase extracellularly whose production increased almost five times on addition of galactose in the culture medium. Production of cellulase has been found optimal at pH 5.5, 37 degrees C and 175 rpm speed using environmental orbital shaker. The cellulase has been purified using ultrafiltration and Sephadex G-200 column chromatography. The native molecular weight of the enzyme is found to be 33,000 +/- 2000 using Sephadex G-200 gel filtration chromatography. The subunit molecular weight (33,000 +/- 2,000) indicate monomeric nature of the enzyme. The enzyme showed Michaelis Menten kinetics exhibiting Km approximately 1.7 +/- 0.1 mg/ml for CMC. The enzyme activity got inhibited by heavy metals viz. Hg+2 and Ag+2.
Biochem Mol Biol Int 1998 Jul
PMID:Production and purification of an extracellular cellulase from Bacillus brevis VS-1. 967 45


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