Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1991 Dec;173(24):7956–7962. doi: 10.1128/jb.173.24.7956-7962.1991

Characterization of endoglucanase A from Clostridium cellulolyticum.

H P Fierobe 1, C Gaudin 1, A Belaich 1, M Loutfi 1, E Faure 1, C Bagnara 1, D Baty 1, J P Belaich 1
PMCID: PMC212590  PMID: 1744052

Abstract

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.

Full text

PDF
7956

Images in this article

7959FIG. 2
on p.7959

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Béguin P., Cornet P., Aubert J. P. Sequence of a cellulase gene of the thermophilic bacterium Clostridium thermocellum. J Bacteriol. 1985 Apr;162(1):102–105. doi: 10.1128/jb.162.1.102-105.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Béguin P. Molecular biology of cellulose degradation. Annu Rev Microbiol. 1990;44:219–248. doi: 10.1146/annurev.mi.44.100190.001251. [DOI] [PubMed] [Google Scholar]
  3. Chauvaux S., Beguin P., Aubert J. P., Bhat K. M., Gow L. A., Wood T. M., Bairoch A. Calcium-binding affinity and calcium-enhanced activity of Clostridium thermocellum endoglucanase D. Biochem J. 1990 Jan 1;265(1):261–265. doi: 10.1042/bj2650261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Durrant A. J., Hall J., Hazlewood G. P., Gilbert H. J. The non-catalytic C-terminal region of endoglucanase E from Clostridium thermocellum contains a cellulose-binding domain. Biochem J. 1991 Jan 15;273(Pt 2):289–293. doi: 10.1042/bj2730289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Faure E., Bagnara C., Belaich A., Belaich J. P. Cloning and expression of two cellulase genes of Clostridium cellulolyticum in Escherichia coli. Gene. 1988 May 15;65(1):51–58. doi: 10.1016/0378-1119(88)90416-7. [DOI] [PubMed] [Google Scholar]
  6. Faure E., Belaich A., Bagnara C., Gaudin C., Belaich J. P. Sequence analysis of the Clostridium cellulolyticum endoglucanase-A-encoding gene, celCCA. Gene. 1989 Dec 7;84(1):39–46. doi: 10.1016/0378-1119(89)90137-6. [DOI] [PubMed] [Google Scholar]
  7. Fürste J. P., Pansegrau W., Frank R., Blöcker H., Scholz P., Bagdasarian M., Lanka E. Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene. 1986;48(1):119–131. doi: 10.1016/0378-1119(86)90358-6. [DOI] [PubMed] [Google Scholar]
  8. Giallo J., Gaudin C., Belaich J. P. Metabolism and Solubilization of Cellulose by Clostridium cellulolyticum H10. Appl Environ Microbiol. 1985 May;49(5):1216–1221. doi: 10.1128/aem.49.5.1216-1221.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Giallo J., Gaudin C., Belaich J. P., Petitdemange E., Caillet-Mangin F. Metabolism of glucose and cellobiose by cellulolytic mesophilic Clostridium sp. strain H10. Appl Environ Microbiol. 1983 Mar;45(3):843–849. doi: 10.1128/aem.45.3.843-849.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gilkes N. R., Warren R. A., Miller R. C., Jr, Kilburn D. G. Precise excision of the cellulose binding domains from two Cellulomonas fimi cellulases by a homologous protease and the effect on catalysis. J Biol Chem. 1988 Jul 25;263(21):10401–10407. [PubMed] [Google Scholar]
  11. Grépinet O., Chebrou M. C., Béguin P. Nucleotide sequence and deletion analysis of the xylanase gene (xynZ) of Clostridium thermocellum. J Bacteriol. 1988 Oct;170(10):4582–4588. doi: 10.1128/jb.170.10.4582-4588.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hall J., Hazlewood G. P., Barker P. J., Gilbert H. J. Conserved reiterated domains in Clostridium thermocellum endoglucanases are not essential for catalytic activity. Gene. 1988 Sep 15;69(1):29–38. doi: 10.1016/0378-1119(88)90375-7. [DOI] [PubMed] [Google Scholar]
  13. Henrissat B., Claeyssens M., Tomme P., Lemesle L., Mornon J. P. Cellulase families revealed by hydrophobic cluster analysis. Gene. 1989 Sep 1;81(1):83–95. doi: 10.1016/0378-1119(89)90339-9. [DOI] [PubMed] [Google Scholar]
  14. Kramer B., Kramer W., Fritz H. J. Different base/base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch-repair system of E. coli. Cell. 1984 Oct;38(3):879–887. doi: 10.1016/0092-8674(84)90283-6. [DOI] [PubMed] [Google Scholar]
  15. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  16. PARK J. T., JOHNSON M. J. A submicrodetermination of glucose. J Biol Chem. 1949 Nov;181(1):149–151. [PubMed] [Google Scholar]
  17. Pilz I., Schwarz E., Kilburn D. G., Miller R. C., Jr, Warren R. A., Gilkes N. R. The tertiary structure of a bacterial cellulase determined by small-angle X-ray-scattering analysis. Biochem J. 1990 Oct 1;271(1):277–280. doi: 10.1042/bj2710277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Tomme P., Van Tilbeurgh H., Pettersson G., Van Damme J., Vandekerckhove J., Knowles J., Teeri T., Claeyssens M. Studies of the cellulolytic system of Trichoderma reesei QM 9414. Analysis of domain function in two cellobiohydrolases by limited proteolysis. Eur J Biochem. 1988 Jan 4;170(3):575–581. doi: 10.1111/j.1432-1033.1988.tb13736.x. [DOI] [PubMed] [Google Scholar]
  19. Wain-Hobson S., Sonigo P., Danos O., Cole S., Alizon M. Nucleotide sequence of the AIDS virus, LAV. Cell. 1985 Jan;40(1):9–17. doi: 10.1016/0092-8674(85)90303-4. [DOI] [PubMed] [Google Scholar]
  20. Yagüe E., Béguin P., Aubert J. P. Nucleotide sequence and deletion analysis of the cellulase-encoding gene celH of Clostridium thermocellum. Gene. 1990 Apr 30;89(1):61–67. doi: 10.1016/0378-1119(90)90206-7. [DOI] [PubMed] [Google Scholar]
  21. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES