Nucleotide sequence and characteristics of endoglucanase gene engB from Clostridium cellulovorans

Foong, Frances; Hamamoto, Toshiro; Shoseyov, Oded; Doi, Roy H.

doi:10.1099/00221287-137-7-1729

Cited by 71 publications

(53 citation statements)

References 24 publications

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“…They were very similar except that EngD had about four-times-greaterp-nitrophenyl 1-1,4-cellobiosidase activity, indicating the presence of cellobiosidase activity as originally reported (14). The purified enzymes retained the substrate specificities of the original clones (6,13,14). The specific activity of EngD was about 80 times greater than that of the unpurified periplasmic fraction of the E. coli clone, and the specific activity of EngB2 purified protein was about 70 times greater than that of the periplasmic fraction of the pC2-engB E. coli clone (6).…”

Section: Resultssupporting

confidence: 53%

“…The purified enzymes retained the substrate specificities of the original clones (6,13,14). The specific activity of EngD was about 80 times greater than that of the unpurified periplasmic fraction of the E. coli clone, and the specific activity of EngB2 purified protein was about 70 times greater than that of the periplasmic fraction of the pC2-engB E. coli clone (6). Isoelectric focusing showed that the isoelectric point of EngD was around 8.8 and that of EngB2 was 7.0 (data not shown).…”

Section: Resultsmentioning

confidence: 91%

“…The template used for the insert of pET-D was pEQ52V (14). The template for pET-B2 was pC2-engB (6). The (35).…”

Section: Methodsmentioning

confidence: 99%

“…It was found that the specificity of C. cellulovorans endoglucanases EngB and EngD was conferred by the NH2-terminal domain and that the COOH-terminal domains of both genes had homology to enzymes from other species of cellulolytic bacteria (6,13). Thus, it would also be interesting to compare our cloned endoglucanases with each other as well as with those of other species in terms of their characteristics and cellulose affinities.…”

mentioning

confidence: 99%

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Characterization and comparison of Clostridium cellulovorans endoglucanases-xylanases EngB and EngD hyperexpressed in Escherichia coli

Foong

Doi

1992

J Bacteriol

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By the use of a T7 expression system, endoglucanases-xylanases EngB and EngD from Clostridium ceflulovorans were hyperexpressed and purified from Escherichia coli. The two enzymes demonstrated both endoglucanase and xylanase activities. The substrate specificities of both endoglucanases were similar except that EngD had four-times-greater p-nitrophenyl 13-1,4-cellobiosidase activity. The two proteins were very homologous (80%o) up to the Pro-Thr-Thr region which divided the protein into -NH2-and -COOH-terminals. The -COOH-region of EngB has high homology to the endoglucanases and a xylanase from Clostridium thermocelum and to an endoglucanase from Clostridium cellulolyticum and did not show strong binding to cellulose (Avicel). However, the -COOH-region of EngD, which had homology to the cellulose-binding domains of Celulomonas fimi exo-and endoglucanases and to Pseudomonas fluorescens endoglucanase, demonstrated binding ability to cellulose even when the domain was fused to the N-terminal domain of EngB. By probing the Avicel-purified cellulase complex (F8) with anti-EngB and anti-EngD antibodies, both EngB and EngD were shown to be present on the cellulase complex of C. cellulovorans. Many proteins homologous to EngB and EngD were also present on the complex.Cellulose degradation is an important part of the biological recycling of carbon. Since the initial investigation of the mechanism of cellulose degradation more than 30 years ago, much progress has been achieved, especially with aerobic fungal cellulases. The degradative process can be different for different species, especially between anaerobic and aerobic organisms. With cellulolytic organisms such as Tichoderma reesei, Neocallismastix frontalis, Pseudomonas fluorescens, Cellulomonas fimi, Fibrobacter succinogenes, and Ruminococcus flavefaciens, it is not certain whether the enzymes essential for the degradation of crystalline cellulose function individually or whether they are present on the cell or substrate surface as a complex (1,15,20). There is evidence that in Clostridium thernocellum as well as in Clostridium strain C7, the enzymes are present in a complex, termed the "cellulosome" (3, 18,19,22,23). There is evidence that the enzymes present in Clostndium cellulovorans are also present in a complex. It has been shown that the native cellulase complex of C. cellulovorans is a multiprotein complex of large molecular mass. The enzymic proteins are probably organized on a nonenzymic scaffolding protein which has been cloned and sequenced (25,27,29). The aim of our investigation was to find out whether our cloned enzymes were part of the cellulase complex.Many cellulolytic enzymes have been found to contain a catalytic domain and a binding domain (5, 8-10, 16, 31, 34). It was found that the specificity of C. cellulovorans endoglucanases EngB and EngD was conferred by the NH2-terminal domain and that the COOH-terminal domains of both genes had homology to enzymes from other species of cellulolytic bacteria (6, 13). Thus, it would also be interesting t...

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Section: Resultssupporting

confidence: 53%

Section: Resultsmentioning

confidence: 91%

“…The template used for the insert of pET-D was pEQ52V (14). The template for pET-B2 was pC2-engB (6). The (35).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Characterization and comparison of Clostridium cellulovorans endoglucanases-xylanases EngB and EngD hyperexpressed in Escherichia coli

Foong

Doi

1992

J Bacteriol

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“…Notably, several members of subfamily A4 have previously been reported to act on more than one substrate. For instance, GH5 proteins from Prevotella ruminicola (AAC36862.1) and Clostridium cellulovorans (AAA23231.1) have been reported to act on glucan as well as xylan substrates, although detailed biochemical characterization or structural information for these enzymes is not available (30,31). The most thoroughly characterized GH5 enzyme from subfamily A4 is the thermostable enzyme, Cel5A_Tma (AAD36816.1), from Thermotoga maritima (32).…”

Section: Building a High Quality Phylogenetic Tree For Gh5-thementioning

confidence: 99%

Tracing Determinants of Dual Substrate Specificity in Glycoside Hydrolase Family 5

Chen

Friedland

Pereira

et al. 2012

Journal of Biological Chemistry

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Background: Glycoside hydrolase family 5 (GH5) comprises enzymes with a wide range of activities critical for the deconstruction of lignocellulose. Results: Concurrent glucan and mannan specificity in over 70 members of GH5 can be ascribed to a conserved active site motif. Conclusion: Single domain multispecific hydrolases are widely prevalent. Significance: This finding has potential applications in improved enzyme mixture design or microbes engineered for consolidated bioprocessing of lignocellulose.

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Species-specificity of the cohesin-dockerin interaction betweenClostridium thermocellum andClostridium cellulolyticum: Prediction of specificity determinants of the dockerin domain

et al. 1997

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The cross-species specificity of the cohesin-dockerin interaction, which defines the incorporation of the enzymatic subunits into the cellulosome complex, has been investigated. Cohesin-containing segments from the cellulosomes of two different species, Clostridium thermocellum and Clostridium cellulolyticum, were allowed to interact with cellulosomal (dockerin-containing) enzymes from each species. In both cases, the cohesin domain of one bacterium interacted with enzymes from its own cellulosome in a calcium-dependent manner, but the same cohesin failed to recognize enzymes from the other species. Thus, in the case of these two bacteria, the cohesin-dockerin interaction seems to be species-specific. Based on intra- and cross-species sequence comparisons among the different dockerins together with their known specificities, we tender a prediction as to the amino-acid residues critical to recognition of the cohesins. The suspected residues were narrowed down to only four, which comprise a repeated pair located within the calcium-binding motif of two duplicated sequences, characteristic of the dockerin domain. According to the proposed model, these four residues do not participate in the binding of calcium per se; instead, they appear to serve as recognition codes in promoting interaction with the cohesin surface.

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Nucleotide sequence and characteristics of endoglucanase gene engB from Clostridium cellulovorans

Cited by 71 publications

References 24 publications

Characterization and comparison of Clostridium cellulovorans endoglucanases-xylanases EngB and EngD hyperexpressed in Escherichia coli

Characterization and comparison of Clostridium cellulovorans endoglucanases-xylanases EngB and EngD hyperexpressed in Escherichia coli

Tracing Determinants of Dual Substrate Specificity in Glycoside Hydrolase Family 5

Species-specificity of the cohesin-dockerin interaction betweenClostridium thermocellum andClostridium cellulolyticum: Prediction of specificity determinants of the dockerin domain

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