Separation and characterization of the complexes constituting the cellulolytic enzyme system of Clostridium thermocellum

Hon-Nami, Koyu; Coughlan, Michael P.; Hon-nami, Hiromi; Ljungdahl, Lars G.

doi:10.1007/bf00413021

Cited by 61 publications

(42 citation statements)

References 19 publications

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“…The majority of OlpAcontaining material could be adsorbed to cellulose. These properties coincide with those described for the "polycellulosomes" (13) and, in fact, CipA was found to be associated with the high-molecular-mass, cellulose-binding fractions containing OlpA. However, this does not mean that OlpA is directly associated with polycellulosomes.…”

Section: Discussionsupporting

confidence: 84%

Subcellular localization of Clostridium thermocellum ORF3p, a protein carrying a receptor for the docking sequence borne by the catalytic components of the cellulosome

et al. 1994

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The ORF3 gene of Clostridium thermocellum encodes a polypeptide (ORF3p) which contains a receptor domain for the docking sequence borne by the catalytic subunits of the cellulosome and a triplicated domain related to some bacterial cell surface proteins. It was thus surmised that ORF3p is a surface protein. In this study, this hypothesis was confirmed. Subcellular fractionation, Western blotting (immunoblotting), and electron microscopy of immunocytochemically labeled cells indicated that ORF3p produced by C. thermocellum was located in the outer surface layer of the bacterium. This layer appeared to consist of a soft matrix shedding off particulate fragments. Nonsedimenting ORF3p derived from sonicated cells was associated with high-molecular-mass fractions (> 20 MDa), probably corresponding to fragments of the outer cell layer. The same high-molecular-mass fractions also contained the cellulosomal marker CipA. Contrary to CipA, however, ORF3p was not associated with 2- to 4-MDa fractions corresponding to individual cellulosomes, and a significant fraction of ORF3p failed to bind to cellulose. It is proposed that ORF3 and ORF3p be renamed olpA and OlpA, respectively (for outer layer protein).

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Section: Discussionsupporting

confidence: 84%

Subcellular localization of Clostridium thermocellum ORF3p, a protein carrying a receptor for the docking sequence borne by the catalytic components of the cellulosome

et al. 1994

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“…Anaerobic microorganisms possess complexed cellulase systems with architecture distinct from the noncomplexed systems of aerobes (11)(12)(13). In cell-free experiments involving complexed cellulase systems, cellobiose and glucose have been observed to accumulate (14,15), consistent with a hydrolysis mechanism similar to that for noncomplexed systems. No evidence has been presented that the primary product of cellulose hydrolysis utilized by anaerobic microorganisms is other than cellobiose.…”

mentioning

confidence: 76%

Cellulose utilization by Clostridium thermocellum : Bioenergetics and hydrolysis product assimilation

Zhang

Lynd

2005

Proc. Natl. Acad. Sci. U.S.A.

220

173

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The bioenergetics of cellulose utilization by Clostridium thermocellum was investigated. Cell yield and maintenance parameters, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}Y_{X/{\mathrm{ATP}}}^{{\mathrm{True}}}=16.44\end{equation*}\end{document} g cell/mol ATP and m = 3.27 mmol ATP/g cell per hour, were obtained from cellobiose-grown chemostats, and it was shown that one ATP is required per glucan transported. Experimentally determined values for \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}G_{{\mathrm{ATP}}}^{P-T}\end{equation*}\end{document} (ATP from phosphorolytic β-glucan cleavage minus ATP for substrate transport, mol ATP/mol hexose) from chemostats fed β-glucans with degree of polymerization (DP) 2-6 agreed well with the predicted value of (n-1)/n (n = mean cellodextrin DP assimilated). A mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}G_{{\mathrm{ATP}}}^{P-T}\end{equation*}\end{document} value of 0.52 ± 0.06 was calculated for cellulose-grown chemostat cultures, corresponding to n = 4.20 ± 0.46. Determination of intracellular β-glucan radioactivity resulting from 14C-labeled substrates showed that uptake is different for cellulose and cellobiose (G2). For 14C-cellobiose, radioactivity was greatest for G2; substantially smaller but measurable for G1, G3, and G4; undetectable for G5 and G6; and n was ≈2. For 14C-cellulose, radioactivity was greatest for G5; lower but substantial for G6, G2, and G1; very low for G3 and G4; and n was ≈4. These results indicate that: (i) C. thermocellum hydrolyzes cellulose by a different mode of action from the classical mechanism involving solubilization by cellobiohydrolase; (ii) bioenergetic benefits specific to growth on cellulose are realized, resulting from the efficiency of oligosaccharide uptake combined with intracellular phosphorolytic cleavage of β-glucosidic bonds; and (iii) these benefits exceed the bioenergetic cost of cellulase synthesis, supporting the feasibility of anaerobic biotechnological processing of cellulosic biomass without added saccharolytic enzymes

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“…Researchers at the University of Georgia (81,131) found even larger aggregates, of ca. 108 ϫ 10 6 Da (polycellulosomes).…”

Section: Cellulosome Structurementioning

confidence: 99%

“…This is the reason that the cellulosome can be dissociated by mild conditions if EDTA is present (25,37,(352)(353)(354). EDTA inhibits cellulosomal activity due to its ability to chelate Ca 2ϩ (131,137,139). Ca 2ϩ is tightly bound in the cellulosome once it is taken up (58).…”

Section: Cohesins and Dockerinsmentioning

confidence: 99%

Cellulase, Clostridia, and Ethanol

Demain

Newcomb

2005

Microbiol Mol Biol Rev

810

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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Separation and characterization of the complexes constituting the cellulolytic enzyme system of Clostridium thermocellum

Cited by 61 publications

References 19 publications

Subcellular localization of Clostridium thermocellum ORF3p, a protein carrying a receptor for the docking sequence borne by the catalytic components of the cellulosome

Subcellular localization of Clostridium thermocellum ORF3p, a protein carrying a receptor for the docking sequence borne by the catalytic components of the cellulosome

Cellulose utilization by Clostridium thermocellum : Bioenergetics and hydrolysis product assimilation

Cellulase, Clostridia, and Ethanol

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