Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Effect of Multiple Copies of Cohesins on Cellulase and Hemicellulase Activities of Clostridium cellulovorans Mini-cellulosomes

  • Cha, Jae-Ho (Section of Molecular and Cellular Biology, University of California) ;
  • Matsuoka, Satoshi (Section of Molecular and Cellular Biology, University of California) ;
  • Chan, Helen (Section of Molecular and Cellular Biology, University of California) ;
  • Yukawa, Hideaki (Research Institute of Innovative Technology for the Earth) ;
  • Inui, Masayuki (Research Institute of Innovative Technology for the Earth) ;
  • Doi, Roy H. (Section of Molecular and Cellular Biology, University of California)
  • Published : 2007.11.30
Download PDF
⟨ Previous Next ⟩

Abstract

Cellulosomes in Clostridium cellulovorans are assembled by the interaction between the repeated cohesin domains of a scaffolding protein (CbpA) and the dockerin domain of enzyme components. In this study, we determined the synergistic effects on cellulosic and hemicellulosic substrates by three different recombinant mini-cellulosomes containing either endoglucanase EngB or endoxylanase XynA bound to mini-CbpA with one cohesin domain (mini-CbpAl), two cohesins (mini-CbpA12), or four cohesins (mini-CbpAl234). The assembly of EngB or XynA with mini-CbpA increased the activity against carboxymethyl cellulose, acid-swollen cellulose, Avicel, xylan, and com fiber 1.1-1.8-fold compared with that for the corresponding enzyme alone. A most distinct improvement was shown with com fiber, a natural substrate containing xylan, arabinan, and cellulose. However, there was little difference in activity between the three different mini-cellulosomes when the cellulosomal enzyme concentration was held constant regardless of the copy number of cohesins in the cellulosome. A synergistic effect was observed when the enzyme concentration was increased to be proportional to the number of cohesins in the mini-cellulosome. The highest degree of synergy was observed with mini-CbpAl234 (1.8-fold) and then mini-CbpAl2 (1.3-fold), and the lowest synergy was observed with mini-CbpAl (1.2-fold) when Avicel was used as the substrate. As the copy number of cohesin was increased, there was more synergy. These results indicate that the clustering effect (physical enzyme proximity) of the enzyme within the mini-cellulosome is one of the important factors for efficient degradation of plant cell walls.

Keywords

References

  1. Bayer, E. A., E. Morag, and R. Lamed. 1994. The cellulosome -- a treasure-trove for biotechnology. Trends Biotechnol. 12: 379-386 https://doi.org/10.1016/0167-7799(94)90039-6
  2. Boraston, A. B., B. W. McLean, J. M. Kormos, M. Alam, N. R. Gilkes, C.A. Haynes, P. Tomme, D. G. Kilburn, and R. A. Warren. 1999. Carbohydrate-binding modules: Diversity of structure and function, pp. 202-211. In H. J. Gilbert, G. J. Davies, B. Henrissat, and B. Svensson (eds.), Recent Advances in Carbohydrate Bioengineering. The Royal Society of Chemistry, Cambridge, United Kingdom
  3. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  4. Cho, H.-Y., H. Yukawa, M. Inui, R. H. Doi, and S.-L. Wong. 2004. Production of minicellulosomes from Clostridium cellulovorans in Bacillus subtilis WB800. Appl. Environ. Microbiol. 70: 5704-5707 https://doi.org/10.1128/AEM.70.9.5704-5707.2004
  5. Doi, R. H. and A. Kosugi. 2004. Cellulosomes: Plant-cellwall- degrading enzyme complexes. Nat. Rev. Microbiol. 2: 541-551 https://doi.org/10.1038/nrmicro925
  6. Dygert, S., L. H. Li, D. Florida, and J. A. Thoma. 1965. Determination of reducing sugar with improved precision. Anal. Biochem. 13: 367-374 https://doi.org/10.1016/0003-2697(65)90327-1
  7. Gerngross, U. T., M. P. Romaniec, T. Kobayashi, N. S. Huskisson, and A. L. Demain. 1993. Sequencing of a Clostridium thermocellum gene (cipA) encoding the cellulosomal SL-protein reveals an unusual degree of internal homology. Mol. Microbiol. 8: 325-334 https://doi.org/10.1111/j.1365-2958.1993.tb01576.x
  8. Goldstein, M. A., M. Takagi, S. Hashida, O. Shoseyov, R. H. Doi, and I. H. Segel. 1993. Characterization of the cellulosebinding domain of the Clostridium cellulovorans cellulosebinding protein A. J. Bacteriol. 175: 5762-5768 https://doi.org/10.1128/jb.175.18.5762-5768.1993
  9. Ichiishi, A., S. Sheweita, and R. H. Doi. 1998. Characterization of EngF from Clostridium cellulovorans and identification of a novel cellulose binding domain. Appl. Environ. Microbiol. 64: 1086-1090
  10. Kataeva, I., G. Guglielmi, and P. Béguin. 1997. Interaction between Clostridium thermocellum endoglucanase CelD and polypeptides derived from the cellulosome-integrating protein CipA: Stoichiometry and cellulolytic activity of the complexes. Biochem. J. 326: 617-624 https://doi.org/10.1042/bj3260617
  11. Kosugi, A., Y. Amano, K. Murashima, and R. H. Doi. 2004. Hydrophilic domains of scaffolding protein CbpA promote glycosyl hydrolase activity and localization of cellulosomes to the cell surface of Clostridium cellulovorans. J. Bacteriol. 186: 6351-6359 https://doi.org/10.1128/JB.186.19.6351-6359.2004
  12. Kosugi, A., K. Murashima, and R. H. Doi. 2002. Characterization of non-cellulosomal subunits, AfrA and BgaA from Clostridium cellulovorans, that cooperate with the cellulosome in plant cell wall degradation. J. Bacteriol. 184: 6859-6865 https://doi.org/10.1128/JB.184.24.6859-6865.2002
  13. Koukiekolo, R., H.-Y. Cho, A. Kosugi, M. Inui, H. Yukawa, and R. H. Doi. 2005. Degradation of corn fiber by Clostridium cellulovorans cellulases and hemicellulases and contribution of scaffolding protein CbpA. Appl. Environ. Microbiol. 71: 3504-3511 https://doi.org/10.1128/AEM.71.7.3504-3511.2005
  14. Kusuma, K., G. H. Chon, J.-S. Lee, J. Kongkiattikajorn, K. Ratanakhanokchai, K. L. Kyu, J. H. Lee, M. S. Roh, Y. Y. Choi, H. Park and Y. S. Lee. 2006. Hydrolysis of agricultural residues and kraft pulps by xylanolytic enzymes from alkaliphilic Bacillus sp. strain BK. J. Microbiol. Biotechnol. 16: 1255-1261
  15. Lamed, R., E. Setter, and E. A. Bayer. 1983. Characterization of a cellulose-binding, cellulase-containing complex in Clostridium thermocellum. J. Bacteriol. 156: 828-836
  16. Lee, Y. E. and P. O. Lim. 2004. Purification and characterization of two thermostable xylanases from Paenibacillus sp. DG- 22. J. Microbiol. Biotechnol. 14: 1014-1021
  17. Morag, E., E. A. Bayer, and R. Lamed. 1990. Relationship of cellulosomal and noncellulosomal xylanases of Clostridium thermocellum to cellulose-degrading enzymes. J. Bacteriol. 172: 6098-6105 https://doi.org/10.1128/jb.172.10.6098-6105.1990
  18. Morag, E., A. Lapidot, D. Govorko, R. Lamed, M. Wilchek, E. A. Bayer, and Y. Shoham. 1995. Expression, purification, and characterization of the cellulose-binding domain of the scaffoldin subunit from the cellulosome of Clostridium thermocellum. Appl. Environ. Microbiol. 61: 1980-1986
  19. Murashima, K., A. Kosugi, and R. H. Doi. 2002. Synergistic effects on crystalline cellulose degradation between cellulosomal cellulases from Clostridium cellulovorans. J. Bacteriol. 184: 5088-5095 https://doi.org/10.1128/JB.184.18.5088-5095.2002
  20. Murashima, K., C. L. Chen, A. Kosugi, Y. Tamaru, R. H. Doi, and S.-L. Wong. 2002. Heterologous production of Clostridium cellulovorans engB, using protease-deficient Bacillus subtilis, and preparation of active recombinant cellulosomes. J. Bacteriol. 184: 76-81 https://doi.org/10.1128/JB.184.1.76-81.2002
  21. Murashima, K., A. Kosugi, and R. H. Doi. 2003. Synergistic effects of cellulosomal xylanase and cellulases from Clostridium cellulovorans on plant cell wall degradation. J. Bacteriol. 185: 1518-1524 https://doi.org/10.1128/JB.185.5.1518-1524.2003
  22. Patthra, P., G. H. Chon, K. Ratanakhanokchai, K. L. Kyu, O.-H. Jhee, J. Kang, W. H. Kim, K.-M. Choi, G.-S. Park, J.-S. Lee, H. Park, M. S. Rho, and Y.-S. Lee. 2006. Selection of multienzyme complex-producing bacteria under aerobic cultivation. J. Microbiol. Biotechnol. 16: 1269-1275
  23. Salamitou, S., O. Raynaud, M. Coughlan, P. Beguin, and J.-P. Aubert. 1994. Recognition specificity of the duplicated segments present in Clostridium thermocellum endoglucanase CelD and in the cellulosome-integrating protein CipA. J. Bacteriol. 176: 2822-2827 https://doi.org/10.1128/jb.176.10.2822-2827.1994
  24. Shoseyov, O., M. Takagi, M. A. Goldstein, and R. H. Doi. 1992. Primary sequence analysis of Clostridium cellulovorans cellulose binding protein A (CbpA). Proc. Natl. Acad. Sci. USA 89: 3483-3487
  25. Stragier, P., C. Bonamy, and C. Karmazyn-Campelli. 1988. Processing of a sporulation sigma factor in Bacillus subtilis: How morphological structure could control gene expression. Cell 52: 697-704 https://doi.org/10.1016/0092-8674(88)90407-2
  26. Takagi, M., S. Hashida, M. A. Goldstein, and R. H. Doi. 1993. The hydrophobic repeated domain of the Clostridium cellulovorans cellulose-binding protein (CbpA) has specific interactions with endoglucanases. J. Bacteriol. 175: 7119-7122 https://doi.org/10.1128/jb.175.21.7119-7122.1993
  27. Tachaapaikoon, C., Y. S. Lee, K. Rantanakhanokchai, S. Pinitglang, K. L. Kyu, M. S. Rho, and S.-K. Lee. 2006. Purification and characterization of two endoxylanases from an alkaliphilic Bacillus halodurans C-1. J. Microbiol. Biotechnol. 16: 613-618
  28. Tamaru, Y. and R. H. Doi. 2001. Pectate lyase A, an enzymatic subunit of the Clostridium cellulovorans cellulosome. Proc. Natl. Acad. Sci. USA 98: 4125-4129
  29. Wu, X.-C., W. Lee, L. Tran, and S.-L. Wong. 1991. Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases. J. Bacteriol. 173: 4952-4958 https://doi.org/10.1128/jb.173.16.4952-4958.1991
  30. Wu, S.-C., J. C. Yeung, Y. Duan, R. Ye, S. J. Szarka, H. R. Habibi, and S.-L. Wong. 2002. Functional production and characterization of a fibrin-specific single-chain antibody fragment from Bacillus subtilis: Effects of molecular chaperones and a wall-bound protease on antibody fragment production. Appl. Environ. Microbiol. 68: 3261-3269 https://doi.org/10.1128/AEM.68.7.3261-3269.2002