Characterization of the subunits in an apparently homogeneous subpopulation of Clostridium thermocellum cellulosomes

Ali, Bassam R.; Romaniec, Marek P.M.; Hazlewood, Geoffrey P.; Freedman, Robert B.

doi:10.1016/0141-0229(94)00118-b

Cited by 39 publications

(26 citation statements)

References 29 publications

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“…Whether the putative repressor Orf4 is involved in the regulation of expression of licA and/or other genes remains to and elucidated. The Lic16A enzyme was found to be secreted and to stay partially cell-bound, but not in the cellulosome: no activity was detected in zymograms of purified cellulosomes from strain F7 and the type strain (data not shown; Ali et al, 1995). The native protein was isolated from the cell surface with a newly described fractionation procedure using GuHCl which would also allow the isolation of other extracellular, cell-bound proteins.…”

Section: Discussionmentioning

confidence: 99%

Lic16A of Clostridium thermocellum, a non-cellulosomal, highly complex endo-β-1,3-glucanase bound to the outer cell surface

Fuchs¹,

Zverlov

Velikodvorskaya

et al. 2003

Microbiology

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Clostridium thermocellum produces one major b-1,3-glucanase. Genomic DNA fragments containing the gene were cloned from two strains, DSM1237T (6848 bp) and F7 (9766 bp).Overlapping sequences were 99?9 % identical. The nucleotide sequences contained reading frames for a putative transposase, endo-b-1,3-1,4-glucanase CelC, a putative transcription regulator of the LacI type, b-1,3-glucanase Lic16A and a putative membrane protein. The licA genes of both strains encoded an identical protein of 1324 aa with a calculated molecular mass of 148 kDa. Lic16A is an unusually complex protein consisting of a leader peptide, a threefold repeat of an S-layer homologous module (SLH), an unknown module, a catalytic module of glycosyl hydrolase family 16 and a fourfold repeat of a carbohydrate-binding module of family CBM4a. The recombinant Lic16A protein was characterized as an endo-1,3(4)-b-glucanase with a specific activity of 2680 and 340 U mg 21 and a K m of 0?94 and 2?1 mg ml 21 towards barley b-glucan and laminarin, respectively. It was specific for b-glucans containing b-1,3-linkages with an optimum temperature of 70˚C at pH 6?0. The N-terminal SLH modules were cleaved from the protein as well in Escherichia coli as in C. thermocellum, but nevertheless bound tightly to the rest of the protein. Lic16A was located on the cell surface from which it could be purified after fractionated solubilization. Its inducible production allowed C. thermocellum to grow on b-1,3-or b-1,3-1,4-glucan.

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Section: Discussionmentioning

confidence: 99%

Lic16A of Clostridium thermocellum, a non-cellulosomal, highly complex endo-β-1,3-glucanase bound to the outer cell surface

Fuchs¹,

Zverlov

Velikodvorskaya

et al. 2003

Microbiology

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“…The organism was found to break down xylan but could not use the resulting xylose or xylose oligomers. Another strain of C. thermocellum (YM4) requires para-aminobenzoic acid, vitamin B 12 , vitamin B 6 , and folic acid (237).…”

Section: Strains and Mediamentioning

confidence: 99%

Cellulase, Clostridia, and Ethanol

Demain

Newcomb

2005

Microbiol Mol Biol Rev

799

579

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SUMMARY Biomass conversion to ethanol as a liquid fuel by the thermophilic and anaerobic clostridia offers a potential partial solution to the problem of the world's dependence on petroleum for energy. Coculture of a cellulolytic strain and a saccharolytic strain of Clostridium on agricultural resources, as well as on urban and industrial cellulosic wastes, is a promising approach to an alternate energy source from an economic viewpoint. This review discusses the need for such a process, the cellulases of clostridia, their presence in extracellular complexes or organelles (the cellulosomes), the binding of the cellulosomes to cellulose and to the cell surface, cellulase genetics, regulation of their synthesis, cocultures, ethanol tolerance, and metabolic pathway engineering for maximizing ethanol yield.

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“…The recombinant proteins bound on the column were eluted with elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4) using a linear gradient (10-500 mM imidazole). After removal of imidazole by dialysis against 50 mM 2-morpholinoethanesulfonic acid (MES)-NaOH buffer (pH 7.0), the purified proteins were treated with thrombin protease (10 units per mg of protein) at 19 C for 16 h to remove the TF and six-His tag fused to their N-terminals. To purify the recombinant proteins from the digested peptide fragments, the mixtures were fractionated again with a HiTrap chelating HP column precharged with Ni 2þ , after the mixtures were dialyzed against the binding buffer.…”

Section: )mentioning

confidence: 99%

“…After overnight incubation, one plaque having mannanase activity was recognized by the formation of a clear halo on a red background after staining with 0.1% Congo red and destaining with 1 M NaCl. 19) The positive plaque isolated was subjected to in vivo excision to excise the pBluescript SK(À) phagemid containing the cloned insert from the ZAPII vector with ExAssist helper phage (Stratagene). The plasmid obtained was named pMANE, and was transfected into E. coli SOLR.…”

Section: )mentioning

confidence: 99%

Cloning and Characterization of a β-1,4-Mannanase 5C Possessing a Family 27 Carbohydrate-Binding Module from a Marine Bacterium,Vibriosp. Strain MA-138

Tanaka

Umemoto

Okamura

et al. 2009

Bioscience, Biotechnology, and Biochemistry

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Characterization of the subunits in an apparently homogeneous subpopulation of Clostridium thermocellum cellulosomes

Cited by 39 publications

References 29 publications

Lic16A of Clostridium thermocellum, a non-cellulosomal, highly complex endo-β-1,3-glucanase bound to the outer cell surface

Lic16A of Clostridium thermocellum, a non-cellulosomal, highly complex endo-β-1,3-glucanase bound to the outer cell surface

Cellulase, Clostridia, and Ethanol

Cloning and Characterization of a β-1,4-Mannanase 5C Possessing a Family 27 Carbohydrate-Binding Module from a Marine Bacterium,Vibriosp. Strain MA-138

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