Characterization of engF, a gene for a non-cellulosomal Clostridium cellulovorans endoglucanase

Sheweita, Salah A.; Ichiishi, Akihiko; Park, Jae-Seon; Liu, Chichi; Malburg, Laercio M.; Doi, Roy H.

doi:10.1016/s0378-1119(96)00544-6

Cited by 20 publications

(15 citation statements)

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“…These genes represent major subunits (cbpA-exgS and engE) (8,20,29,34), one of the hemicellulase genes (xynA) (18), and the pectate lyase gene (pelA) (33). Two noncellulosomal genes, engF (27) and the ␣-L-arabinofuranosidase gene arfA (16), were also tested as control-endoglucanase genes. A semiquantitative measure of the level of cbpA mRNA, using DIG-labeled probes and RNA isolated at different times during cell growth, was obtained by Northern blot analysis ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Regulation of Expression of Cellulosomal Cellulase and Hemicellulase Genes in Clostridium cellulovorans

et al. 2003

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The regulation of expression of the genes encoding the cellulases and hemicellulases of Clostridium cellulovorans was studied at the mRNA level with cells grown under various culture conditions. A basic pattern of gene expression and of relative expression levels was obtained from cells grown in media containing poly-, di-or monomeric sugars. The cellulase (cbpA and engE) and hemicellulase (xynA) genes were coordinately expressed in medium containing cellobiose or cellulose. Growth in the presence of cellulose, xylan, and pectin gave rise to abundant expression of most genes (cbpA-exgS, engH, hbpA, manA, engM, engE, xynA, and/or pelA) studied. Moderate expression of cbpA, engH, manA, engE, and xynA was observed when cellobiose or fructose was used as the carbon source. Low levels of mRNA from cbpA, manA, engE, and xynA were observed with cells grown in lactose, mannose, and locust bean gum, and very little or no expression of cbpA, engH, manA, engE, and xynA was detected in glucose-, galactose-, maltose-, and sucrose-grown cells. The cbpA-exgS and engE genes were most frequently expressed under all conditions studied, whereas expression of xynA and pelA was more specifically induced at higher levels in xylan-or pectin-containing medium, respectively. Expression of the genes (cbpA, hbpA, manA, engM, and engE) was not observed in the presence of most soluble di-or monosaccharides such as glucose. These results support the hypotheses that there is coordinate expression of some cellulases and hemicellulases, that a catabolite repression type of mechanism regulates cellulase expression in rapidly growing cells, and that the presence of hemicelluloses has an effect on cellulose utilization by the cell.

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Section: Resultsmentioning

confidence: 99%

Regulation of Expression of Cellulosomal Cellulase and Hemicellulase Genes in Clostridium cellulovorans

et al. 2003

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“…Several cellulases from fungi [26] and bacteria [27,28] show optimal hydrolytic activities under similar conditions. Cel14b22 is active over a wide range of pH, retaining 40% of its activity between pH 4.5 and pH 9.5, and it is stable even at pH 10.0.The Cel14b22 endoglucanase was only partially inhibited when incubated in 1 m M EDTA, suggesting that Cel14b22 does not absolutely require divalent cations to degrade CMC.…”

Section: Discussionmentioning

confidence: 99%

Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene

et al. 2012

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BackgroundInterest in cellulose degrading enzymes has increased in recent years due to the expansion of the cellulosic biofuel industry. The rumen is a highly adapted environment for the degradation of cellulose and a promising source of enzymes for industrial use. To identify cellulase enzymes that may be of such use we have undertaken a functional metagenomic screen to identify cellulase enzymes from the bacterial community in the rumen of a grass-hay fed dairy cow.ResultsTwenty five clones specifying cellulose activity were identified. Subcloning and sequence analysis of a subset of these hydrolase-positive clones identified 10 endoglucanase genes. Preliminary characterization of the encoded cellulases was carried out using crude extracts of each of the subclones. Zymogram analysis using carboxymethylcellulose as a substrate showed a single positive band for each subclone, confirming that only one functional cellulase gene was present in each. One cellulase gene, designated Cel14b22, was expressed at a high level in Escherichia coli and purified for further characterization. The purified recombinant enzyme showed optimal activity at pH 6.0 and 50°C. It was stable over a broad pH range, from pH 4.0 to 10.0. The activity was significantly enhanced by Mn2+ and dramatically reduced by Fe3+ or Cu2+. The enzyme hydrolyzed a wide range of beta-1,3-, and beta-1,4-linked polysaccharides, with varying activities. Activities toward microcrystalline cellulose and filter paper were relatively high, while the highest activity was toward Oat Gum.ConclusionThe present study shows that a functional metagenomic approach can be used to isolate previously uncharacterized cellulases from the rumen environment.

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“…The formation of these complexes is necessary for the efficient degradation of crystalline cellulose (2,3,7,31,33). However, some noncellulosomal enzymes of C. cellulovorans, such as EngD (14), EngF (32), ArfA (19), and BgaA (19), are not bound to this multienzyme complex. These proteins appear to be part of an ancillary enzyme system for the hydrolysis of polysaccharides.…”

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Regulation of Expression of Cellulosomes and Noncellulosomal (Hemi)Cellulolytic Enzymes in Clostridium cellulovorans during Growth on Different Carbon Sources

et al. 2004

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Cellulosomes and noncellulosomal (hemi)cellulolytic enzymes are produced by Clostridium cellulovorans to degrade plant cell walls. To understand their synergistic relationship, changes in mRNA and protein expression in cellulosomes and noncellulosomal (hemi)cellulolytic enzymes (hereafter called noncellulosomal enzymes) of cultures grown on cellobiose, cellulose, pectin, xylan, and corn fiber or mixtures thereof were examined. Cellulase expression, favored particularly by the presence of Avicel, was found with all substrates. Comparison of cellulosome and noncellulosomal enzymes showed that expression profiles were strongly affected by the carbon source. High xylanase or pectate lyase expression was observed when C. cellulovorans was grown on xylan or pectin, respectively. Mixed carbon substrates (cellulose-pectin-xylan mixture or corn fiber) induced a wider variety of enzymes than a single carbon source, such as cellobiose, pectin, or xylan. Cellulosomal proteome profiles were more affected by the carbon source than the noncellulosomal enzymes. Transcription and protein analyses revealed that cellulosomes and noncellulosomal enzymes were expressed simultaneously on mixed carbon sources, but their degree of inducibility varied when the substrate was either cellulose or cellobiose. Cellulosomes and noncellulosomal enzymes had synergistic activity on various carbon substrates. These results indicated that expression of plant cell wall-degrading enzymes is highly influenced by the available carbon source and that synergy between cellulosomes and noncellulosomal enzymes contribute to plant cell wall degradation.

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Characterization of engF, a gene for a non-cellulosomal Clostridium cellulovorans endoglucanase

Cited by 20 publications

References 17 publications

Regulation of Expression of Cellulosomal Cellulase and Hemicellulase Genes in Clostridium cellulovorans

Regulation of Expression of Cellulosomal Cellulase and Hemicellulase Genes in Clostridium cellulovorans

Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene

Regulation of Expression of Cellulosomes and Noncellulosomal (Hemi)Cellulolytic Enzymes in Clostridium cellulovorans during Growth on Different Carbon Sources

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