Skip to main content
SpringerLink
Log in

Carboxymethylcellulase and avicelase activities from a cellulolytic Clostridium strain A11

  • Published:
Current Microbiology Aims and scope Submit manuscript

Abstract

The extracellular cellulase enzyme system of Clostridium A11 was fractionated by affinity chromatography on Avicel: 80% of the initial carboxymethylcellulase (CMCase) activity was adhered. This cellulase system was a multicomponent aggregate. Several CMCase activities were detected, but the major protein P1 had no detectable activity. Adhered and unadhered cellulases showed CMCase activity with the highest specific activity in Avicel-adhered fraction. However, only afhered fractions could degrade Avicel. Thus, efficiency of the enzymatic hydrolysis of Avicel was related to the cellulase-adhesion capacity. Carboxymethylcellulase and Avicelase activities were studied with the extracellular enzyme system and cloned cellulases. Genomic libraries from Clostridium A11 were constructed with DNA from this Clostridium, and a new gene cel1 was isolated. The gene(s) product(s) from cel1 exhibited CMCase and p-nitrophenylcellobiosidase (pNPCbase) activities. This cloned cellulase adhered to cellulose. Synergism between “adhered enzyme system” and cloned endoglucanases was observed on Avicel degradation. Conversely, no synergism was observed on CMC hydrolysis. Addition of cloned endoglucanase to cellulase complex led to increase of the Vmax without significant K m variation. Cloned endoglucanases can be added to cellulase complexes to efficiently hydrolyze cellulose.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Literature Cited

  1. Benoit L, Cailliez C, Petitdemange E, Gitton J (1992) Isolation of cellulolytic mesophilic clostridia from a municipal solid waste digestor. Microb Ecol 23:117–125

    Google Scholar 

  2. Cailliez C, Benoit L, Thirion JP, Petitdemange H (1992) Characterization of 10 mesophilic cellulolytic clostridia isolated from a municipal solid waste digestor. Curr Microbiol 25:105–112

    Google Scholar 

  3. Cailliez C, Benoit L, Gelhaye E, Petitdemange H, Raval G (1993) Solubilization of cellulose by mesophilic cellulolytic clostridia isolated from a municipal solid waste digestor. Bioresour Technol 43:77–83

    Google Scholar 

  4. Deshpande MV, Petterson LG, Eriksson KE (1984) An assay for selective determination of exo-1,4-glucanase in a mixture of cellulolytic enzymes. Anal Biochem 138:481–487

    Google Scholar 

  5. Dower WJ, Miller JF, Ragsdale CW (1988) High efficiency transformation of Escherichia coli by high voltage electroporation. Nucleic Acids Res 16:6127–6145

    Google Scholar 

  6. Faure E, Belaich A, Bagnara C, Belaich JP (1989) Sequence analysis of the Clostridium cellulolyticum endoglucanase-a-encoding gene celCCA. Gene 85:39–46

    Google Scholar 

  7. Fierobe HP, Gaudin C, Belaich A, Loufti M, Faure E, Bagnara C, Baty D, Belaich JP (1991) Characterization of endoglucanase A from Clostridium cellulolyticum. J Bacteriol 24:7956–7962

    Google Scholar 

  8. Foong F, Hamamoto T, Shoseyov O, Doi RH (1991) Nucleotide sequence and characteristics of endoglucanase gene engB from Clostridium cellulovorans. J Gen Microbiol 137:1729–1736

    Google Scholar 

  9. Gelhaye E, Benoit L, Petitdemange H, Gay R (1993a) Adhesive properties of five mesophilic cellulolytic Clostridia isolated from the same biotope. FEMS Microbiol Ecol 102:67–73

    Google Scholar 

  10. Gelhaye E, Gehin A, Benoit L, Petitdemange H (1993b) Effect of cellobiose on cellulose colonization by a mesophilic cellulolytic Clostridium (strain C401). J Gen Microbiol 139:2819–2824

    Google Scholar 

  11. Greener A (1990) Escherichia coli SURE strain clone “unclonable”. DNA Strategies Mol Biol 1:5–9

    Google Scholar 

  12. Kadam SK, Demain AL (1989) Addition of cloned β-glucosidase enhances the degradation of crystalline cellulose by the Clostridium thermocellum cellulase complex. Biochem Biophys Res Commun 161:706–711

    Google Scholar 

  13. Kitto GB (1969) Intra and extramitochondrial malate dehydrogenase from chicken and tuna heart. Methods Enzymol 13:106–116

    Google Scholar 

  14. Klyosov AA (1990) Trends in biochemistry and enzymology of cellulose degradation. Biochemistry 29:10577–10585

    Google Scholar 

  15. Kremer S, Wood PM (1992) Continuous monitoring of cellulase action on microcrystalline cellulose. Appl Microbiol Biotechnol 37:750–755

    Google Scholar 

  16. Lamed R, Kenig R, Setter, Bayer EA (1985) Major characteristics of the cellulolytic system of Clostridium thermocellum coincide with those of the purified cellulosome. Enzyme Microbiol Technol 7:37–41

    Google Scholar 

  17. Lamed R, Kenig R, Morag-Morgenstem E, Calzada JF, De Micheo F, Bayer EA (1991) Efficient solubilization by a combined cellulosome-β-glucosidase system. Appl Biochem Biotechnol 27:173–183

    Google Scholar 

  18. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    Google Scholar 

  19. Madarro A, Pena JL, Valles S, Gay R, Flors A (1991) Partial purification of the cellulases from Clostridium cellulolyticum H10. J Chem Technol Biotechnol 52:393–406

    Google Scholar 

  20. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugars. Anal Biochem 31:426–428

    Google Scholar 

  21. Miller GL, Blum R, Glennon W, Burton AL (1960) Measurement of carboxymethylcellulase activity. Anal Chem 138:481–487

    Google Scholar 

  22. Neu HC, Heppel LA (1965) The release of enzymes from Escherichia coli by osmotic shock and during the formation of sphaeroplasts. J Biol Chem 240:3685–3692

    Google Scholar 

  23. Nisman B (1968) Techniques for demonstrating DNA-dependent protein synthesis. Methods Enzymol 12 B:794–820

    Google Scholar 

  24. Nolte A, Mayer F (1989) Localization and immunological characterization of the cellulolytic enzyme system in Clostridium thermocellum. FEMS Microbiol Lett 61:65–72

    Google Scholar 

  25. Park JT, Jonhson MS (1949) A submicrodetermination of glucose. J Biol Chem 181:265–275

    Google Scholar 

  26. Perez-Martinez G, Gonzalez-Candelas L, Polaina J, Flors A (1988) Expression of an endoglucanase gene from Clostridium cellulolyticum in Escherichia coli. J Ind Microbiol 3:365–371

    Google Scholar 

  27. Petitdemange E, Caillet F, Giallo J, Gaudin C (1984) Clostridium cellulolyticum sp. nov., a cellulolytic mesophilic species from decayed grass. Int J Syst Bacteriol 34:155–159

    Google Scholar 

  28. Quiviger B, Franche C, Lutfalla G, Rice D, Haselkorn R, Elmerich C (1982) Cloning of nitrogen fixation (nif) gene cluster of Azospirillum brasilense. Biochemie 64:495–502

    Google Scholar 

  29. Romaniec MP, Fauth U, Kobayashi T, Huskinsson NS, Barker PJ, Demain AL (1992) Purification and characterization of a new endoglucanase from Clostridium themocellum. Biochem J 283:69–73

    Google Scholar 

  30. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd ed. Cold Spring Harbor, N.Y.: Cold Springer Harbor Laboratory Press

    Google Scholar 

  31. Sebald M (1993) Genetics and molecular biology of anaerobic bacteria. New York, Heidelberg, Berlin: Springer-Verlag

    Google Scholar 

  32. Shoseyov O, Doï RH (1990) Essential 170-kDa subunit for degradation of crystalline cellulose by Clostridium cellulovorans cellulase. Proc Natl Acad Sci USA 87:2192–2195

    Google Scholar 

  33. Theather RM, Wood PJ (1982) Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from bovine rumen. Appl Environ Microbiol 43:777–780

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratoire de Chimie Biologique I, Université de Nancy I, BP 239, 54506, Vandœuvre-lès-Nancy Cédex, France

    L. Benoit, C. Cailliez, A. Gehin, G. Raval & H. Petitdemange

  2. Département de Microbiologie CHUS Faculté de Médecine, Université de Sherbrooke, J1H-5N4, P.Q., Canada

    J. Thirion

Authors
  1. L. Benoit
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. C. Cailliez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. Gehin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. Thirion
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. G. Raval
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. H. Petitdemange
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Benoit, L., Cailliez, C., Gehin, A. et al. Carboxymethylcellulase and avicelase activities from a cellulolytic Clostridium strain A11. Current Microbiology 30, 305–312 (1995). https://doi.org/10.1007/BF00295506

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00295506

Keywords

Search

Navigation