Characterization of endoglucanase A from Clostridium cellulolyticum

A construction was carried out to obtain a high level of expression in Escherichia coli of the gene celCCA, coding for the endoglucanase A from Clostridium cellulolyticum (EGCCA). The enzyme was purified in two forms with different molecular weights, 51,000 and 44,000. The smaller protein was probably the result of proteolysis, although great care was taken to prevent this process from occurring. Evidence was found for the loss of the conserved reiterated domains which are characteristic of C. thermocellum and C. cellulolyticum cellulases. The two forms were extensively studied, and it was demonstrated that although they had the same pH and temperature optima, they differed in their catalytic properties. The truncated protein gave the more efficient catalytic parameters on carboxymethyl cellulose and showed improved endoglucanase characteristics, whereas the intact enzyme showed truer cellulase characteristics. The possible role of clostridial reiterated domains in the hydrolytic activity toward crystalline cellulose is discussed.

[1]  D. Kilburn,et al.  Precise excision of the cellulose binding domains from two Cellulomonas fimi cellulases by a homologous protease and the effect on catalysis. , 1988, The Journal of biological chemistry.

[2]  J. Giallo,et al.  Metabolism of glucose and cellobiose by cellulolytic mesophilic Clostridium sp. strain H10 , 1983, Applied and environmental microbiology.

[3]  Olivier Danos,et al.  Nucleotide sequence of the AIDS virus, LAV , 1985, Cell.

[4]  H. van Tilbeurgh,et al.  Limited proteolysis of the cellobiohydrolase I from Trichoderma reesei , 1986 .

[5]  J. Millet,et al.  Genes and Proteins Involved in Cellulose and Xylan Degradation by Clostridium thermocellum , 1993 .

[6]  A. Demain,et al.  Molecular cloning of a gene for a thermostable β-glucosidase from Clostridium thermocellum into Escherichia coli , 1988 .

[7]  Christian Gaudin,et al.  Metabolism and Solubilization of Cellulose by Clostridium cellulolyticum H10 , 1985, Applied and environmental microbiology.

[8]  A Bairoch,et al.  Calcium-binding affinity and calcium-enhanced activity of Clostridium thermocellum endoglucanase D. , 1990, The Biochemical journal.

[9]  J. Hall,et al.  Conserved reiterated domains in Clostridium thermocellum endoglucanases are not essential for catalytic activity. , 1988, Gene.

[10]  C. Gaudin,et al.  Genes and Proteins Involved in Cellulose Degradation by Mesophilic Clostridia , 1993 .

[11]  H. Fritz,et al.  Different base/base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch-repair system of E. coli , 1984, Cell.

[12]  J. Aubert,et al.  Sequence of a cellulase gene of the thermophilic bacterium Clostridium thermocellum , 1985, Journal of bacteriology.

[13]  E. Faure,et al.  Sequence analysis of the Clostridium cellulolyticum endoglucanase-A-encoding gene, celCCA. , 1989, Gene.

[14]  P Béguin,et al.  Molecular biology of cellulose degradation. , 1990, Annual review of microbiology.

[15]  J. Hall,et al.  The non-catalytic C-terminal region of endoglucanase E from Clostridium thermocellum contains a cellulose-binding domain. , 1991, The Biochemical journal.

[16]  B Henrissat,et al.  Cellulase families revealed by hydrophobic cluster analysis. , 1989, Gene.

[17]  M. J. Johnson,et al.  A submicrodetermination of glucose. , 1949, The Journal of biological chemistry.

[18]  C. Yanisch-Perron,et al.  Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. , 1985, Gene.

[19]  E. Faure,et al.  Cloning and expression of two cellulase genes of Clostridium cellulolyticum in Escherichia coli. , 1988, Gene.

[20]  J. Aubert,et al.  Nucleotide sequence and deletion analysis of the cellulase-encoding gene celH of Clostridium thermocellum. , 1990, Gene.

[21]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[22]  H. van Tilbeurgh,et al.  Studies of the cellulolytic system of Trichoderma reesei QM 9414. Binding of small ligands to the 1,4-beta-glucan cellobiohydrolase II and influence of glucose on their affinity. , 1989, European journal of biochemistry.

[23]  J. Giallo,et al.  Clostridium cellulolyticum sp. nov., a cellulolytic, mesophilic species from decayed grass , 1984 .

[24]  O. Grépinet,et al.  Nucleotide sequence and deletion analysis of the xylanase gene (xynZ) of Clostridium thermocellum , 1988, Journal of bacteriology.

[25]  H. Blöcker,et al.  Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. , 1986, Gene.