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

doi:10.1016/0922-338X(93)90189-F

Journal of Fermentation and Bioengineering

Volume 76, Issue 4, 1993, Pages 251-256

Journal of Fermentat...

https://doi.org/10.1016/0922-338X(93)90189-F Get rights and content

Abstract

The nucleotide sequence of a new endoglucanase gene (celM) of Clostridium thermocellum was determined. The structural gene contains an open reading frame of 978 by starting with an ATG codon and terminating in a TGA stop codon. The deduced amino acid sequence of endoglucanase M (CeIM) contains 325 amino acids (MW 35,186) and its N-terminal amino acid sequence is identical to that of the purified enzyme. The codon usage of the gene is similar to that of other endoglucanase genes in the same organism. The endoglucanase was purified from Escherichia coli with a 200-fold increase in specific activity. The optimum pH and temperature values of the enzyme are 5.5 to 6.5 and 60°C respectively for carboxymethylcellulose (CMC) degradation.

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Among the characterized proteins, Pcal_0842 exhibited almost similar identity to a metallopeptidase and a cellulase. A 46.45% identity was found with a characterized metallopeptidase from Pyrococcus horikoshii (accession # O59196) [21] and a 46.46% identity with a cellulase (CelM) from Clostridium thermocellum (accession # L13461) [22]. An alignment of amino acid sequences of Pcal_0842 and CelM is shown in Fig. 1.
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Lic16A is, in addition, able to degrade ßβ-1,3-glucan (Fuchs et al., 2003). CelM was reported to have endo-glucanase activity (Kobayashi et al., 1993); however, its role remains doubtful because the sequence is not homologous to any other endo-glucanase but to aminopeptidases. The soluble enzyme component Cel9I has a high degree of similarity to Cel9Z of C. stercorarium (in structure and sequence homology), which is also a soluble cellulase.
This chapter focuses on the most prevalent polysaccharides present in biomass: starch, cellulose, and hemicellulose—the latter two of which are especially difficult substrates to degrade. Anaerobic bacteria, among them primarily the clostridia, are an excellent source for hydrolytic enzymes that are able to hydrolyze polysaccharides in biomass to fermentable sugars. The chapter also discusses the unique strategies of the clostridia to cope with these substrate problems and highlights the features of some of the extracellular enzymes produced by these bacteria to degrade or hydrolyze biopolymers such as starch or cellulose. Some of these enzymes or enzyme systems are unique among micro-organisms. The enormous potential of clostridia as producers of industrially important enzymes is obvious. In addition, genetic tools for clostridia have been developed for custom engineering of new production strains. Thus it seems to be possible now to engineer an enzyme with optimal features for a given purpose or even to create a special Clostridium species that is able to convert cheap, renewable biomass into desired valuable products.
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^§: Permanent address: Tochigi Research Laboratories, Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi 321-34, Japan.

^†: The other authors dedicate this paper to the memory of Marek Romaniec who died at the untimely age of 31 on Nov. 13, 1992. The gene is named celM and the enzyme endoglucanase M (CelM) in Marek's honor. His entire scientific life was devoted to the study of the cellulase complex of C. thermocellum.

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

Abstract

FEMS Microbiol. Lett.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Enzyme Microb. Technol.

Gene

Gene

Res. Microbiol.

Gene

Biochimie

Biochimie

Gene

Gene

Enzyme Microb. Technol.

Anal. Biochem.

Gene

J. Mol. Biol.

Cell

J. Biol. Chem.

J. Biotechnol.

The cellulo- some—A discrete cell surface organelle of Clostridium thermocellum which exhibits separate antigenic, cellulose-binding and various cellulolytic activities

Macromolecular organization of the cellulolytic enzyme complex of Clostridium thermocellum as revealed by electron microscopy

Appl. Environ. Microbiol.

Cloning and expression in Escherichia coli of thermophilic Clostridium sp. FI genes related to cellulose hydrolysis

Agric. Biol. Chem.

Nucleotide sequence and deletion analysis of the xylanase gene (xynZ) of Clostridium thermocellum

J. Bacteriol.

Identification of three distinct Clostridium thermocellum xylanase genes by molecular cloning

Arch. Microbiol.

Structure of the Clostridium thermocellum gene licB and the encoded β-1,3-1,4 glucanase; a catalytic region homologous to Bacillus lichenase joined to the reiterated domain of clostridial cellulases

Eur. J. Biochem.

Characterization of two β-glucosidase genes from Clostridium thermocellum

Biotechnol. Lett.

Molecular cloning of Clostridium thermocellum DNA and the expression of further novel endo-β-1,4-glucanase genes in Escherichia coli

J. Gen. Microbiol.

Sequence of a cellulase gene of the thermophilic bacterium Clostridium thermocellum

J. Bacteriol.

Sequence of the cellulase gene of Clostridium thermocellum coding for endoglucanase B

Nucleic Acids Res.

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