Antonie van Leeuwenhoek

, Volume 66, Issue 4, pp 319–326 | Cite as

Characterization of production and enzyme properties of an endo-β-1,4-glucanase fromBacillus subtilis CK-2 isolated from compost soil

  • Kari Aa
  • Ragnar Flengsrud
  • Viggo Lindahl
  • Arne Tronsmo
Research Articles


Bacillus subtilis CK-2, isolated from garden organic waste compost, was found to have high hydrolytic activity against carboxymethylcellulose (CMC) due to the secretion of an endo-β-1,4-glucanase. Enzyme production was related to the sporulation process, and was regulated by the concentration of readily metabolizable carbohydrate in growth medium. Enzyme production did not require CMC or other cellulose containing materials. The endo-β-1,4-glucanase activity was optimal at pH 5.6–5.8 and at 65 MoC, and achieved thermal stability up to 55 MoC. The activity was inhibited by Hg2+. The purified enzyme gave a single band corresponding to a MW of 35.5 kDa on SDS-PAGE, while the Sephadex G-75 chromatography revealed a molecular weight of the active enzyme around 70 kDa, indicating a dimeric form of the active enzyme. The enzyme activity was irreversibly inhibited by SDS. Native PAGE and IEF revealed three different isoelectric forms of the enzyme, all with an identical N-terminal amino-acid sequence.

Key words

Bacillus subtilis endo-β-1,4-glucanase 







sodium dodecyl sulfate


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  1. Au K-S & Chan K-Y (1987) Purification and properties of the endo-β-1,4-glucanase fromBacillus subtilis. J. Gen. Microbiol. 133: 2155–2162Google Scholar
  2. Béguin P (1983) Detection of cellulase activity in polyacrylamide gels using Congo red-stained agar replicas. Anal. Biochem. 131: 333–336Google Scholar
  3. Chan K-Y & Au K-S (1987) Studies on cellulase production by aBacillus subtilis. Antonie van Leeuwenhoek 53: 125–136Google Scholar
  4. Cornelis P, Digneffe C & Willemot K (1982) Cloning and expression of aBacillus coagulans amylase gene inEscherichia coli. Mol. Gen. Genet. 186: 507–511Google Scholar
  5. Deshpande MW, Petterson LG & Eriksson K-E (1988) Selective assay for exo-1,4-β-glucanases. In: Wood WA & Kellogg ST (Eds) Methods in Enzymology, Vol. 160: Biomass. Part A: Cellulose and Hemicellulose (pp 126–130) Academic Press, San DiegoGoogle Scholar
  6. Doi RH (1989) Sporulation and germination. In: Harwood CL (Ed)Bacillus, Biotechnology Handbooks, Vol. 2. Plenum Press, New YorkGoogle Scholar
  7. Eriksson K-EL, Blanchette RA & Ander P (1990) Microbial and enzymatic degradation of wood and wood components. Springer Verlag, Berlin HeidelbergGoogle Scholar
  8. Ghose TK (1987) Measurement of cellulase activities. Pure & Appl. Chem. 59: 257–268Google Scholar
  9. Hirano H (1989) Microsequence analysis of winged bean seed proteins electroblotted from two-dimensional gel. J. Protein Chemistry 8: 115–130Google Scholar
  10. Horikoshi K & Fukumori F (1988) Modification and expression of alkaline cellulase genes of alkalophilicBacillus strains. In: Aubert JP, Béguin P & Millet J (Eds) Biochemistry and Genetics of Cellulose Degradation. FEMS Symposium No. 43 (pp 203–217). Academic Press, San DiegoGoogle Scholar
  11. Horikoshi K, Nakao M, Kurono Y & Sashihara N (1984) Cellulase of an alkalophilicBacillus strain isolated from soil. Can. J. Microbiol. 30: 774–779Google Scholar
  12. Ito S, Shikata S, Ozaki K, Kawai S, Okamoto K, Inoue S, Takel A, Ohta Y & Satoh T (1989) Alkaline cellulase for laundry detergents: production byBacillus sp. KSM-635 and enzymatic properties. Agric. Biol. Chem. 53: 1275–1281Google Scholar
  13. Koide Y, Nakamura A, Uozumi T & Beppu T (1986) Molecular cloning of a cellulase gene fromBacillus subtilis and its expression inEscherichia coli. Agric. Biol. Chem. 50: 233–237Google Scholar
  14. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685Google Scholar
  15. Matsudaira P (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidine difluoride membranes. J. Biol. Chem. 262: 10035–10038Google Scholar
  16. Okoshi H, Ozaki K, Shikata S, Oshino K, Kawai S & Ito S (1990) Purification and characterization of multiple carboxymethyl cellulases fromBacillus sp. KSM-522. Agric. Biol. Chem. 54: 83–89Google Scholar
  17. Ozaki K & Ito S (1991) Purification and properties of an acid endo-β-1,4-glucanase fromBacillus sp. KSM-330. J. Gen. Microbiol. 137: 41–48Google Scholar
  18. Pétré J, Longin R & Millet J (1981) Purification and properties of an endo-β-1,4-glucanase fromClostridium thermocellum. Biochimie 63: 629–639Google Scholar
  19. Priest FG (1977) Extracellular enzyme synthesis in the genusBacillus. Bacteriol. Rev. 41: 711–753Google Scholar
  20. Robson LM & Chambliss GH (1984) Characterization of the cellulolytic activity of aBacillus isolate. Appl. Environ. Microbiol. 47: 1039–1046Google Scholar
  21. —— (1987) Endo-β-1,4-glucanase gene ofBacillus subtilis DLG. J. Bacteriol. 169: 2017–2025Google Scholar
  22. —— (1989) Cellulases of bacterial origin. Enzyme Microbiol. Technol. 11: 626–644Google Scholar
  23. Ryu DDY & Mandels M (1980) Cellulases: biosynthesis and applications. Enzyme Microbiol. Technol. 2: 91–102Google Scholar
  24. Sharma P, Gupta JK, Vadehra DV & Dube DK (1990) Purification and properties of an endoglucanase from aBacillus isolate. Enzyme Microbiol. Technol. 12: 132–137Google Scholar
  25. Yoshimatsu T, Ozaki K, Shikata S, Ohta Y-I, Koike K, Kawai S & Ito S (1990) Purification and characterization of alkaline endo-β-1,4-glucanases from alkalophilicBacillus sp. KSM-635. J. Gen. Microbiol. 136: 1973–1979Google Scholar

Copyright information

© Kluwer Academic Publishers 1994

Authors and Affiliations

  • Kari Aa
    • 1
  • Ragnar Flengsrud
    • 1
  • Viggo Lindahl
    • 1
  • Arne Tronsmo
    • 1
  1. 1.Department of Biotechnological SciencesAgricultural University of NorwayAsNorway

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