Nucleotide sequence of gene celM encoding a new endoglucanase (CeIM) of Clostridium thermocellum and purification of the enzyme

Kobayashi, Tohru; Romaniec, Marek P.M.; Barker, Paul D.; Gerngross, U. T.; Demain, Arnold L.

doi:10.1016/0922-338x(93)90189-f

Cited by 14 publications

(11 citation statements)

References 53 publications

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“…Therefore, the existence of the repeated sequence in the CelS protein supports an interaction between CelS (Ss) and SL, as has been observed for their synergism in cellulase activity (58,59 On the basis of the following evidence, we concluded that the cloned celS gene codes for the CelS protein previously reported: (i) the calculated molecular weight is in close agreement with the apparent molecular weight of the native CelS protein; (ii) the celS sequence agrees with three peptide sequences determined for the CelS protein, one at the N terminus and the other two near the middle and the C terminus of CelS; and (iii) the celS sequence contains the conserved, duplicated sequence that so far has been found only in the clostridial genes coding for glucanases and that supports the interaction with the SL protein, as previously reported (58,59). We compared the celS sequence with that of the J3 gene (25), cloned by use of antibodies against CelS, and found no global similarities. The calculated molecular weight of the product of the J3 gene (35,186) is much lower than that of CelS.…”

Section: G P T Ksupporting

confidence: 67%

Cloning and DNA sequence of the gene coding for Clostridium thermocellum cellulase Ss (CelS), a major cellulosome component

Wang

Kruus

1993

J Bacteriol

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Clostridium thermocellum ATCC 27405 produces an extracellular cellulase system capable of hydrolyzing crystalline cellulose. The enzyme system involves a multicomponent protein aggregate (the cellulosome) with a total molecular weight in the millions, impeding mechanistic studies. However, two major components of the aggregate, SS (M(r) = 82,000) and SL (M(r) = 250,000), which act synergistically to hydrolyze crystalline cellulose, have been identified (J. H. D. Wu, W. H. Orme-Johnson, and A. L. Demain, Biochemistry 27:1703-1709, 1988). To further study this synergism, we cloned and sequenced the gene (celS) coding for the SS (CelS) protein by using a degenerate, inosine-containing oligonucleotide probe whose sequence was derived from the N-terminal amino acid sequence of the CelS protein. The open reading frame of celS consisted of 2,241 bp encoding 741 amino acid residues. It encoded the N-terminal amino acid sequence and two internal peptide sequences determined for the native CelS protein. A putative ribosome binding site was identified at the 5' end of the gene. A putative signal peptide of 27 amino acid residues was adjacent to the N terminus of the CelS protein. The predicted molecular weight of the secreted protein was 80,670. The celS gene contained a conserved reiterated sequence encoding 24 amino acid residues found in proteins encoded by many other clostridial cel or xyn genes. A palindromic structure was found downstream from the open reading frame. The celS gene is unique among the known cel genes of C. thermocellum. However, it is highly homologous to the partial open reading frame found in C. cellulolyticum and in Caldocellum saccharolyticum, indicating that these genes belong to a new family of cel genes.

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Section: G P T Ksupporting

confidence: 67%

Cloning and DNA sequence of the gene coding for Clostridium thermocellum cellulase Ss (CelS), a major cellulosome component

Wang

Kruus

1993

J Bacteriol

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“…This was the case in our analysis of celM. The only experimental evidence for endoglucanase activity of CelM is evidence for the celM gene in C. thermocellum (24). The Arctic CelM was 29% identical and 48% similar to the C. thermocellum CelM (Table 1).…”

Section: Resultsmentioning

confidence: 60%

Sequence and Expression Analyses of Cytophaga -Like Hydrolases in a Western Arctic Metagenomic Library and the Sargasso Sea

Cottrell

Kirchman

2005

Appl Environ Microbiol

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Sequence analysis of environmental DNA promises to provide new insights into the ecology and biogeochemistry of uncultured marine microbes. In this study we used the Sargasso Sea Whole Genome Sequence (WGS) data set to search for hydrolases used by Cytophaga-like bacteria to degrade biopolymers such as polysaccharides and proteins. Analysis of the Sargasso WGS data for contigs bearing both the 16S rRNA genes of Cytophaga-like bacteria and hydrolase genes revealed a cellulase gene (celM) most similar to the gene found in Cytophaga hutchinsonii. A BLAST search of the entire Sargasso Sea WGS data set indicated that celM was the most abundant cellulase-like gene in the Sargasso Sea. However, the similarity between CelM-like cellulases and peptidases belonging to metalloprotease family M42 led us to question whether CelM is involved in the degradation of polysaccharides or proteins. PCR primers were designed for the celM genes in the Sargasso Sea WGS data set and used to identify celM in a fosmid library constructed with prokaryotic DNA from the western Arctic Ocean. Expression analysis of the Cytophaga-like Arctic CelM, which is 63% identical and 77% similar to CelM in C. hutchinsonii, indicated that there was peptidase activity, whereas cellulase activity was not detected. Our analysis suggests that the celM gene plays a role in the degradation of protein by Cytophagalike bacteria. The abundance of peptidase genes in the Cytophaga-like fosmid clone provides further evidence for the importance of Cytophaga-like bacteria in the degradation of protein in high-molecular-weight dissolved organic matter.

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“…The substrate specificities of the two enzymes were similar. A third new endoglucanase was CelM (162). Its nucleotide sequence indicated a molecular mass of 35 kDa.…”

Section: Genes and Enzymesmentioning

confidence: 99%

“…CelF, the CelS equivalent in C. cellulolyticum, also displays significant activity in reducing the viscosity of a CMC solution (271). Some of the cloned endoglucanase genes are celA (26), celB (105), celC (298), celD (143,326), celE (113), celF (245), celG (188), celH (361), celI (101,123,373), celJ (encoding component S2 of the complex) (1,8), celM (162), celN (373), celQ (9), celT (172), and celX (113).…”

Section: Genes and Enzymesmentioning

confidence: 99%

Cellulase, Clostridia, and Ethanol

Demain

Newcomb

2005

Microbiol Mol Biol Rev

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806

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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Nucleotide sequence of gene celM encoding a new endoglucanase (CeIM) of Clostridium thermocellum and purification of the enzyme

Cited by 14 publications

References 53 publications

Cloning and DNA sequence of the gene coding for Clostridium thermocellum cellulase Ss (CelS), a major cellulosome component

Cloning and DNA sequence of the gene coding for Clostridium thermocellum cellulase Ss (CelS), a major cellulosome component

Sequence and Expression Analyses of Cytophaga -Like Hydrolases in a Western Arctic Metagenomic Library and the Sargasso Sea

Cellulase, Clostridia, and Ethanol

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