A catalogue of Clostridium thermocellum endoglucanase, β-glucosidase and xylanase genes cloned in Escherichia coli

Hazlewood, Geoffrey P.; Romaniec, Marek P.M.; Davidson, Keith; Grépinet, Olivier; Béguin, Pierre; Millet, J; Raynaud, Odette; Aubert, Jean-Paul

doi:10.1111/j.1574-6968.1988.tb03002.x

Cited by 108 publications

(25 citation statements)

References 12 publications

(11 reference statements)

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“…Screening of gene banks based on the detection of catalytically active recombinant proteins led to the cloning of genes encoding 15 endoglucanases, two xylanases [132,133], two /3-glucosidases [134], and one lichenase (/3-1,3-/3-1,4-glucan glucanohydrolase) [135] from C. thermocellum NCIB 10682. Dissociation in the presence of sodium dodecyl sulfate followed by PAGE reveals the presence of at least 14 different types of polypeptides with M r values ranging from 48000 to 210000.…”

Section: The Cellulosome Conceptmentioning

confidence: 99%

The biological degradation of cellulose

Béguin

Aubert

1994

FEMS Microbiology Reviews

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Cellulolytic microorganisms play an important role in the biosphere by recycling cellulose, the most abundant carbohydrate produced by plants. Cellulose is a simple polymer, but it forms insoluble, crystalline microfibrils, which are highly resistant to enzymatic hydrolysis. All organisms known to degrade cellulose efficiently produce a battery of enzymes with different specificities, which act together in synergism. The study of cellulolytic enzymes at the molecular level has revealed some of the features that contribute to their activity. In spite of a considerable diversity, sequence comparisons show that the catalytic cores of cellulases belong to a restricted number of families. Within each family, available data suggest that the various enzymes share a common folding pattern, the same catalytic residues, and the same reaction mechanism, i.e. either single substitution with inversion of configuration or double substitution resulting in retention of the beta-configuration at the anomeric carbon. An increasing number of three-dimensional structures is becoming available for cellulases and xylanases belonging to different families, which will provide paradigms for molecular modeling of related enzymes. In addition to catalytic domains, many cellulolytic enzymes contain domains not involved in catalysis, but participating in substrate binding, multi-enzyme complex formation, or possibly attachment to the cell surface. Presumably, these domains assist in the degradation of crystalline cellulose by preventing the enzymes from being washed off from the surface of the substrate, by focusing hydrolysis on restricted areas in which the substrate is synergistically destabilized by multiple cutting events, and by facilitating recovery of the soluble degradation products by the cellulolytic organism. In most cellulolytic organisms, cellulase synthesis is repressed in the presence of easily metabolized, soluble carbon sources and induced in the presence of cellulose. Induction of cellulases appears to be effected by soluble products generated from cellulose by cellulolytic enzymes synthesized constitutively at a low level. These products are presumably converted into true inducers by transglycosylation reactions. Several applications of cellulases or hemicellulases are being developed for textile, food, and paper pulp processing. These applications are based on the modification of cellulose and hemicellulose by partial hydrolysis. Total hydrolysis of cellulose into glucose, which could be fermented into ethanol, isopropanol or butanol, is not yet economically feasible. However, the need to reduce emissions of greenhouse gases provides an added incentive for the development of processes generating fuels from cellulose, a major renewable carbon source.

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Section: The Cellulosome Conceptmentioning

confidence: 99%

The biological degradation of cellulose

Béguin

Aubert

1994

FEMS Microbiology Reviews

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1,098

330

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“…Investigations have involved sequencing glucosidase genes (Hazlewood et al 1988;Liebl et al 1994;Dan et al 2000), kinetic analysis of mutants (Lawson et al 1998;Ly & Withers 1999;Li et al 2001), studies on the effect of pH on the enzyme kinetics, inhibition studies and selective chemical modification (Lundblad & Noyes 1985;Keresztessy et al 1994a;Skoubas & Georgatsos 1997). An alternative approach is the application of irreversible inhibitors, like the mechanism-based inhibitors (Withers et al 1988(Withers et al , 1990 and the affinity labels.…”

Section: Introductionmentioning

confidence: 98%

Investigation of the active site of the extracellular β-D-glucosidase from Aspergillus carbonarius

Jäger

Kiss

2005

World J Microbiol Biotechnol

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The catalytic amino acid residues of the extracellular b-D-glucosidase (b-D-glucoside glucohydrolase, EC 3.2.1.21) from Aspergillus carbonarius were investigated. The pH dependence curves gave apparent pK values of 2.8 and 5.93 for the free enzyme, and 2.24 and 6.14 for the enzyme-substrate complex using p-nitrophenyl-b-D-glucoside as substrate. Carbodiimide-and Woodward reagent K-mediated chemical modifications suggested that a carboxylate residue, located in the active centre, was fundamental in the catalysis. The pH dependence of inactivation revealed the involvement of a group with pK value of 4.61 in the modification reaction, proving that a carboxylate residue was modified. The A. carbonarius b-glucosidase was irreversibly inactivated by N-bromoacetyl-b-D-glucopyranosylamine. The active site specificity of the inactivation was proved by using the competitive inhibitor p-nitrophenyl-1-thio-b-D-glucopyranoside. pH Dependence studies of inactivation revealed that modification by N-bromoacetyl-b-D-glucopyranosylamine could be directed toward the carboxylate group acting as the catalytic nucleophile, as in the case of the carbodiimide and Woodward reagent K modifications.Abbreviations: pATP-Glc

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“…Similar insertions or deletions were reported by Hazlewood et al in cellulase genes from different strains of C. thermocellum. 22 ) They ascribed this phenomenon to a transposable element that could occur at various loci. Further investigation, however, on the proposed element has not been done.…”

Section: Discussionmentioning

confidence: 99%

Purification and Characterization of Xylanase A fromClostridium stercorariumF-9 and a RecombinantEscherichia coli

Sakka

Kojima

Kondo

et al. 1994

Bioscience, Biotechnology, and Biochemistry

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Xylanase A encoded by the Clostridium stercorarium F-9 xynA gene was purified to homogeneity from a recombinant clone of Escherichia coli. The N-terminal amino acid sequence and molecular weight (54,000) estimated by SDS-PAGE of the purified enzyme were consistent with those deduced from the nucleotide sequence [Biosci. Biotech. Biochem., 57, 273-277 (1993)]. A xylanase was also purified to homogeneity from a culture supernatant of C. stercorarium F-9. Its N-terminal amino acid sequence, molecular weight, and enzymatic properties were quite in agreement with those of the recombinant enzyme, indicating that the xynA gene was predominantly expressed as a xylanase gene in C. stercorarium F-9. The purified enzyme hydrolyzed xylotriose to yield xylobiose and xylose while it was less active toward xylobiose. It was optimally active at 75 degrees C and pH 7.0 Km and Vmax were estimated to be 1.9 mg/ml and 2.8 mumol of xylose equivalent/min/micrograms for oat spelt xylan, respectively.

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A catalogue of Clostridium thermocellum endoglucanase, β-glucosidase and xylanase genes cloned in Escherichia coli

Cited by 108 publications

References 12 publications

The biological degradation of cellulose

The biological degradation of cellulose

Investigation of the active site of the extracellular β-D-glucosidase from Aspergillus carbonarius

Purification and Characterization of Xylanase A fromClostridium stercorariumF-9 and a RecombinantEscherichia coli

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