Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1993 May;59(5):1573–1578. doi: 10.1128/aem.59.5.1573-1578.1993

Biochemical Characterization of a Protease Involved in the Processing of a Streptomyces reticuli Cellulase (Avicelase)

Michael Moormann 1, André Schlochtermeier 1, Hildgund Schrempf 1,*
PMCID: PMC182121  PMID: 16348937

Abstract

A 36-kDa protease from Streptomyces reticuli had recently been shown to be responsible for the in vivo and in vitro processing of the 82-kDa cellulase (Avicelase) Cel-1 from S. reticuli to a 42-kDa truncated enzyme. It was induced only in the presence of Avicel, hydroxyethylcellulose, and xylan. The addition of the nonionic detergent Tween 80 to the culture medium containing Avicel as the carbon source led to a 10-fold increase in extracellular proteolytic activity. The protease, which has an isoelectric point of 3.9, was purified to homogeneity from the culture filtrate by a combination of anion-exchange and hydrophobic-interaction chromatographies and was characterized biochemically. The enzyme hydrolyzed gelatin and the chromogenic substrates Azocoll, Azocasein, and Azoalbumin. Its highest activity was determined between pH 7.0 and 7.7 and at 55°C. The proteolytic activity was inhibited by 1,10-phenanthroline and EDTA; however, no metal ions were detected to be associated with the protein. The protease was stable in the presence of 1 M urea and 0.01 M sodium dodecyl sulfate. The inhibitory effect of alpha-2-macroglobulin indicated an endo-mode of proteolytic cleavage. Studies with lectins and sugar analysis by mass spectroscopy indicated that the cellulase (Avicelase) Cel-1 was neither N nor O glycosylated. Its processing by the protease occurred at temperatures ranging from 30 to 55°C, pH 7.5, in the presence of 2 mM dithiothreitol.

Full text

PDF
1573

Images in this article

1576Image
on p.1576

Selected References

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

  1. Barrett A. J. Protein degradation in health and disease. Introduction: the classification of proteinases. Ciba Found Symp. 1979;(75):1–13. [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Eriksson K. E., Pettersson B. Purification and partial characterization of two acidic proteases from the white-rot fungus Sporotrichum pulverulentum. Eur J Biochem. 1982 Jun;124(3):635–642. doi: 10.1111/j.1432-1033.1982.tb06641.x. [DOI] [PubMed] [Google Scholar]
  5. Gerwig G. J., de Waard P., Kamerling J. P., Vliegenthart J. F., Morgenstern E., Lamed R., Bayer E. A. Novel O-linked carbohydrate chains in the cellulase complex (cellulosome) of Clostridium thermocellum. 3-O-Methyl-N-acetylglucosamine as a constituent of a glycoprotein. J Biol Chem. 1989 Jan 15;264(2):1027–1035. [PubMed] [Google Scholar]
  6. Gilkes N. R., Kilburn D. G., Miller R. C., Jr, Warren R. A. Structural and functional analysis of a bacterial cellulase by proteolysis. J Biol Chem. 1989 Oct 25;264(30):17802–17808. [PubMed] [Google Scholar]
  7. Gilkes N. R., Langsford M. L., Kilburn D. G., Miller R. C., Jr, Warren R. A. Mode of action and substrate specificities of cellulases from cloned bacterial genes. J Biol Chem. 1984 Aug 25;259(16):10455–10459. [PubMed] [Google Scholar]
  8. 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]
  9. Henriksson G., Pettersson G., Johansson G., Ruiz A., Uzcategui E. Cellobiose oxidase from Phanerochaete chrysosporium can be cleaved by papain into two domains. Eur J Biochem. 1991 Feb 26;196(1):101–106. doi: 10.1111/j.1432-1033.1991.tb15791.x. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Langsford M. L., Gilkes N. R., Singh B., Moser B., Miller R. C., Jr, Warren R. A., Kilburn D. G. Glycosylation of bacterial cellulases prevents proteolytic cleavage between functional domains. FEBS Lett. 1987 Dec 10;225(1-2):163–167. doi: 10.1016/0014-5793(87)81150-x. [DOI] [PubMed] [Google Scholar]
  13. Nakayama M., Tomita Y., Suzuki H., Nisizawa K. Partial proteolysis of some cellulase components from Trichoderma viride and the substrate specificity of the modified products. J Biochem. 1976 May;79(5):955–966. doi: 10.1093/oxfordjournals.jbchem.a131163. [DOI] [PubMed] [Google Scholar]
  14. Schlochtermeier A., Niemeyer F., Schrempf H. Biochemical and Electron Microscopic Studies of the Streptomyces reticuli Cellulase (Avicelase) in Its Mycelium-Associated and Extracellular Forms. Appl Environ Microbiol. 1992 Oct;58(10):3240–3248. doi: 10.1128/aem.58.10.3240-3248.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Schlochtermeier A., Walter S., Schröder J., Moorman M., Schrempf H. The gene encoding the cellulase (Avicelase) Cel1 from Streptomyces reticuli and analysis of protein domains. Mol Microbiol. 1992 Dec;6(23):3611–3621. doi: 10.1111/j.1365-2958.1992.tb01797.x. [DOI] [PubMed] [Google Scholar]
  16. Schwarz W. H., Bronnenmeier K., Gräbnitz F., Staudenbauer W. L. Activity staining of cellulases in polyacrylamide gels containing mixed linkage beta-glucans. Anal Biochem. 1987 Jul;164(1):72–77. doi: 10.1016/0003-2697(87)90369-1. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Wachinger G., Bronnenmeier K., Staudenbauer W. L., Schrempf H. Identification of Mycelium-Associated Cellulase from Streptomyces reticuli. Appl Environ Microbiol. 1989 Oct;55(10):2653–2657. doi: 10.1128/aem.55.10.2653-2657.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES