Cloning of Clostridiumcellulovoransendo-1,4-β-glucanase genes

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

doi:10.1016/0006-291x(90)90382-w

Cited by 35 publications

(23 citation statements)

References 9 publications

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“…The hyperexpression of EngD but not of EngB was achieved easily. Also, attempts to purify EngB from 10 liters of cell culture by using the original clone, pC2 (26), which had a 3.2-kb fragment containing the gene in vector pUC19, were unsuccessful. It was also noticed that clones of pC2 and those with the entire engB gene grew very slowly (6).…”

Section: Discussionmentioning

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: Discussionmentioning

confidence: 99%

“…We have cloned four endoglucanase genes (engA, engB, engC [26], and engD [14]) and a cellulose-binding-protein gene, cbpA (27). Since the individual proteins in the cellulase complex are very difficult to separate, our strategy was to clone the genes and hyperproduce and purify each protein separately so that we could study and compare them.…”

mentioning

confidence: 99%

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

Foong

Doi

1992

J Bacteriol

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“…The proteolytic susceptibility of the connecting linker between the carbohydrate binding module (CBM) moiety and the enzyme facilitated isolation of the individual domain, leading to the first CBM isolation of the fungus Trichoderma reesei and the bacterium Cellulomonas fimi (69,194,201). While this model is still controversial, the first C 1 component was cloned from Clostridium cellulovorans and Cellulomonas fimi (49,74,172,173).…”

Section: Introductionmentioning

confidence: 99%

Carbohydrate Binding Modules: Biochemical Properties and Novel Applications

Shoseyov

Shani

Levy

2006

Microbiol Mol Biol Rev

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488

347

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SUMMARY Polysaccharide-degrading microorganisms express a repertoire of hydrolytic enzymes that act in synergy on plant cell wall and other natural polysaccharides to elicit the degradation of often-recalcitrant substrates. These enzymes, particularly those that hydrolyze cellulose and hemicellulose, have a complex molecular architecture comprising discrete modules which are normally joined by relatively unstructured linker sequences. This structure is typically comprised of a catalytic module and one or more carbohydrate binding modules (CBMs) that bind to the polysaccharide. CBMs, by bringing the biocatalyst into intimate and prolonged association with its substrate, allow and promote catalysis. Based on their properties, CBMs are grouped into 43 families that display substantial variation in substrate specificity, along with other properties that make them a gold mine for biotechnologists who seek natural molecular “Velcro” for diverse and unusual applications. In this article, we review recent progress in the field of CBMs and provide an up-to-date summary of the latest developments in CBM applications.

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“…As a result of the single insertion at this position, the nucleotide sequence around this region in EngC is read as Cys-Glu-Gly-Ile-His, instead of Ala-Lys-Gly-Phe-Thr, and the translation is ended immediately downstream of this nucleotide sequence by generation of a stop codon, TAA. However, the molecular mass of EngC expressed in E. coli, was shown to be approximately 70 kDa, 30) a value very close to the mature EG-IV. If the single insertion of the T was not correct for the nucleotide sequence of EngC gene, its deduced amino acid sequence is almost identical to that of the mature EG-IV.…”

Section: Amino Acid Sequence Analysismentioning

confidence: 93%

“…M95180; Shoseyov et al 30)), belonging to cellulase family E2. Especially, the amino acid residues from Ser 26 to Phe 529 are completely identical in the two sequences.…”

Section: Amino Acid Sequence Analysismentioning

confidence: 99%