Skip to main content
SpringerLink
Log in

Biochemical Characterization and Overexpression of an Endo-rhamnogalacturonan Lyase from Penicillium chrysogenum

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Rhamnogalacturonan lyase (PcRGL4A) was purified from the culture supernatant of Penicillium chrysogenum 31B. PcRGL4A optimal activity occurred between pH 7–8 and at 40 °C. Conserved Domain Search analysis identified PcRGL4A as a member of Polysaccharide Lyase family 4. PcRGL4A contains two conserved catalytic and four conserved substrate-binding residues as determined by X-ray crystallography of the Aspergillus aculeatus RG lyase. Recombinant PcRGL4A (rPcRGL4A) expressed in Escherichia coli demonstrated specific activity against rhamnogalacturonan (RG) but not homogalacturonan. Analysis of the RG reaction products by high-performance anion-exchange chromatography revealed that rPcRGL4A cleaved the substrate in an endo-manner and that the major final product was an RG tetrasaccharide with 4-deoxy-4,5-unsaturated galacturonic acid at the nonreducing end. Based on these results, PcRGL4A was classified as an endo-acting RG lyase (EC 4.2.2.23). Divalent cations were not essential for the enzymatic activity of rPcRGL4A, but addition of calcium ions to the reaction mixture increased enzymatic activity. rPcRGL4A demonstrated a preference for RG lacking galactose decoration.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. McNeil, M., Darvill, A. G., Fry, S. C., & Albersheim, P. (1984). Structure and function of the primary cell walls of plants. Annual Review of Biochemistry, 53, 625–663.

    Article  CAS  Google Scholar 

  2. Thakur, B. R., Singh, R. K., & Handa, A. K. (1997). Chemistry and uses of pectin—A review. Critical Reviews in Food Science and Nutrition, 37, 47–73.

    Article  CAS  Google Scholar 

  3. Willats, W. G. T., McCartney, L., Mackie, W., & Knox, J. P. (2001). Pectin: Cell biology and prospects for functional analysis. Plant Molecular Biology, 47, 9–27.

    Article  CAS  Google Scholar 

  4. Voragen, A. G. J., Coenen, G. J., Verhoef, R. P., & Schols, H. (2009). Pectin, a versatile polysaccharide present in plant cell walls. Structural Chemistry, 20, 263–275.

    Article  CAS  Google Scholar 

  5. O’Neil, M. A., Warrenfeltz, D., Kates, K., Pellerin, P., Doco, T., Darvill, A. G., et al. (1996). Rhamnogalacturonan-II, a pectic polysaccharide in the walls of growing plant cell, forms a dimer that is covalently cross-linked by a borate ester. In vitro conditions for the formation and hydrolysis of the dimer. Journal of Biological Chemistry, 271, 22923–22930.

    Article  Google Scholar 

  6. Willats, W. G. T., Knox, P., & Mikkelsen, J. D. (2006). Pectin: New insights into an old polymer are starting to gel. Trends in Food Science & Technology, 17, 97–104.

    Article  CAS  Google Scholar 

  7. Thomassen, L. V., Vigsnæs, L. K., Licht, T. R., Mikkelsen, J. D., & Meyer, A. S. (2011). Maximal release of highly bifidogenic soluble dietary fibers from industrial potato pulp by minimal enzymatic treatment. Applied Microbiology and Biotechnology, 90, 873–884.

    Article  CAS  Google Scholar 

  8. Onumpai, C., Kolida, S., Bonnin, E., & Rastall, R. A. (2011). Microbial utilization and selectivity of pectin fractions with various structures. Applied and Environment Microbiology, 77, 5747–5754.

    Article  CAS  Google Scholar 

  9. Lærke, H. N., Meyer, A. S., Kaack, K. V., & Larsen, T. (2007). Soluble fiber extracted from potato pulp is highly fermentable but has no effect on risk markers of diabetes and cardiovascular disease in Goto-Kakizaki rats. Nutrition Research, 27, 152–160.

    Article  Google Scholar 

  10. Schols, H. A., Geraeds, C. C. J. M., Searle-van Leeuwen, M. F., Kormelink, F. J. M., & Voragen, A. G. J. (1990). Rhamnogalacturonase: a novel enzyme that degrades the hairy regions of pectins. Carbohydrate Research, 206, 105–115.

    Article  CAS  Google Scholar 

  11. Mutter, M., Colquhoun, I. J., Schols, H. A., Beldman, G., & Voragen, A. G. J. (1996). Rhamnogalacturonase B from Aspergillus aculeatus is a rhamnogalacturonan α-L-rhamnopyranosyl-(1->4)-α-d-galactopyranosyluronide lyase. Plant Physiology, 110, 73–77.

    Article  CAS  Google Scholar 

  12. Mutter, M., Colquhoun, I. J., Beldman, G., Schols, H. A., Bakx, E. J., & Voragen, A. G. J. (1998). Characterization of recombinant rhamnogalacturonan α-L-rhamnopyranosyl-(1,4)-α-D-galactopyranosyluronide lyase from Aspergillus aculeatus. An enzyme that fragments rhamnogalacturonan I regions of pectin. Plant Physiology, 117, 141–152.

    Article  CAS  Google Scholar 

  13. Mutter, M., Renard, C. M. G. C., Beldman, G., Schols, H. A., & Voragen, A. G. J. (1998). Mode of action of RG-hydrolase and RG-lyase toward rhamnogalacturonan oligomers. Characterization of degradation products using RG-rhamnohydrolase and RG-galacturonohydrolase. Carbohydrate Research, 311, 155–164.

    Article  CAS  Google Scholar 

  14. Kofod, L. V., Kauppinen, S., Christgau, S., Andersen, L. N., Heldt-Hansen, H. P., Dörreich, K., et al. (1994). Cloning and characterization of two structurally and functionally divergent rhamnogalacturonases from Aspergillus aculeatus. Journal of Biological Chemistry, 269, 29182–29189.

    CAS  Google Scholar 

  15. Laatu, M., & Condemine, G. (2003). Rhamnogalacturonate lyase RhiE is secreted by the out system in Erwinia chrysanthemi. Journal of Bacteriology, 185, 1642–1649.

    Article  CAS  Google Scholar 

  16. Yoshino-Yasuda, S., Karita, S., Kato, M., & Kitamoto, N. (2012). Sequence analysis and heterologous expression of rhamnogalacturonan lyase A gene (AsrglA) from Shoyu Koji Mold, Aspergillus sojae KBN1340. Food Science and Technology Research, 18, 901–909.

    Article  CAS  Google Scholar 

  17. McKie, V. A., Vincken, J. P., van den Voragen, A. G. J., Broek, L. A. M., Stimson, E., & Gilbert, H. J. (2001). A new family of rhamnogalacturonan lyases contains an enzyme that binds to cellulose. Biochemical Journal, 355, 167–177.

    Article  CAS  Google Scholar 

  18. Pagès, S., Valette, O., Abdou, L., Bélaïch, A., & Bélaïch, J. P. (2003). A rhamnogalacturonan lyase in the Clostridium cellulolyticum cellulosome. Journal of Bacteriology, 185, 4727–4733.

    Article  Google Scholar 

  19. Ochiai, A., Yamasaki, M., Itoh, T., Mikami, B., Hashimoto, W., & Murata, K. (2006). Crystallization and preliminary X-ray analysis of the rhamnogalacturonan lyase YesW from Bacillus subtilis strain 168, a member of polysaccharide lyase family 11. Acta Crystallographica Section F, 62, 438–440.

    CAS  Google Scholar 

  20. Ochiai, A., Itoh, T., Kawamata, A., Hashimoto, W., & Murata, K. (2007). Plant cell wall degradation by saprophytic Bacillus subtilis strains: gene clusters responsible for rhamnogalacturonan depolymerization. Applied and Environment Microbiology, 73, 3803–3813.

    Article  CAS  Google Scholar 

  21. Ochiai, A., Itoh, T., Maruyama, Y., Kawamata, A., Mikami, B., Hashimoto, W., et al. (2007). A novel structural fold in polysaccharide lyases: Bacillus subtilis family 11 rhamnogalacturonan lyase YesW with an eight-bladed β-propeller. Journal of Biological Chemistry, 282, 37134–37145.

    Article  CAS  Google Scholar 

  22. Silva, I. R., Larsen, D. M., Meyer, A. S., & Mikkelsen, J. D. (2011). Identification, expression, and characterization of a novel bacterial RGI lyase enzyme for the production of bio-functional fibers. Enyzme and Microbial Technology, 49, 160–166.

    Article  CAS  Google Scholar 

  23. Sakamoto, T., & Thibault, J. F. (2001). An exo-arabinanase of Penicillium chrysogenum able to release arabinobiose from α-1,5-L-arabinan. Applied and Environment Microbiology, 67, 3319–3321.

    Article  CAS  Google Scholar 

  24. Sakamoto, T., Ihara, H., Kozaki, S., & Kawasaki, H. (2003). A cold-adapted endo-arabinanase from Penicillium chrysogenum. Biochimica et Biophysica Acta, 1624, 70–75.

    Article  CAS  Google Scholar 

  25. Sakamoto, T., Ihara, H., Shibano, A., Kasai, N., Inui, H., & Kawasaki, H. (2004). Molecular characterization of a Penicillium chrysogenum exo-1,5-α-L-arabinanase that is structurally distinct from other arabinan-degrading enzymes. FEBS Letters, 560, 199–204.

    Article  CAS  Google Scholar 

  26. Sakamoto, T., Ogura, A., Inui, M., Tokuda, S., Hosokawa, S., Ihara, H., et al. (2011). Identification of a GH62 α-L-arabinofuranosidase specific for arabinoxylan produced by Penicillium chrysogenum. Applied Microbiology and Biotechnology, 90, 137–146.

    Article  CAS  Google Scholar 

  27. Sakamoto, T., Inui, M., Yasui, K., Tokuda, S., Akiyoshi, M., Kobori, Y., et al. (2012). Biochemical characterization and gene expression of two endo-arabinanases from Penicillium chrysogenum 31B. Applied Microbiology and Biotechnology, 93, 1087–1096.

    Article  CAS  Google Scholar 

  28. Sakamoto, T., Inui, M., Yasui, K., Hosokawa, S., & Ihara, H. (2013). Substrate specificity and gene expression of two Penicillium chrysogenum α-L-arabinofuranosidases (AFQ1 and AFS1) belonging to glycoside hydrolase families 51 and 54. Applied Microbiology and Biotechnology, 97, 1121–1130.

    Article  CAS  Google Scholar 

  29. Shinozaki, A., Kawakami, T., Hosokawa, S., & Sakamoto, T. (2014). A novel GH43 α-L-arabinofuranosidase of Penicillium chrysogenum that preferentially degrades single-substituted arabinosyl side chains in arabinan. Enyzme and Microbial Technology, 58–59, 80–86.

    Article  Google Scholar 

  30. Thibault, J. F., Renard, C. M. G. C., Axelos, M. A. V., Roger, P., & Crépeau, M. J. (1993). Studies of the length of homogalacturonic regions in pectins by acid hydrolysis. Carbohydrate Research, 238, 271–286.

    Article  CAS  Google Scholar 

  31. Tanabe, H., Kobayashi, Y., Matuo, Y., Nishi, N., & Wada, F. (1984). Isolation and fundamental properties of endo-pectate lyase pI-isozymes from Erwinia carotovora. Agricultural and Biological Chemistry, 48, 2113–2120.

    Article  CAS  Google Scholar 

  32. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.

    Article  CAS  Google Scholar 

  33. Shevchenko, A., Wilm, M., Vorm, O., & Mann, M. (1996). Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Analytical Chemistry, 68, 850–858.

    Article  CAS  Google Scholar 

  34. Blumenkrantz, N., & Asboe-Hansen, G. (1973). New method for quantitative determination of uronic acids. Analytical Biochemistry, 54, 484–489.

    Article  CAS  Google Scholar 

  35. Van den Berg, M. A., Albang, R., Albermann, K., Badger, J. H., Daran, J. M., Driessen, A. J., et al. (2008). Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum. Nature Biotechnology, 26, 1161–1168.

    Article  CAS  Google Scholar 

  36. Marcet-Houben, M., de la Ballester, A. R., Fuente, B., Harries, E., Marcos, J. F., González-Candelas, L., et al. (2012). Genome sequence of the necrotrophic fungus Penicillium digitatum, the main postharvest pathogen of citrus. BMC Genomics, 13, 646.

    Article  Google Scholar 

  37. Galagan, J. E., Calvo, S. E., Cuomo, C., Ma, L.-J., Wortman, J. R., Batzoglou, S., et al. (2005). Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae. Nature, 438, 1105–1115.

    Article  CAS  Google Scholar 

  38. McDonough, M. A., Kadirvelraj, R., Harris, P., Poulsen, J. C., & Larsen, S. (2004). Rhamnogalacturonan lyase reveals a unique three-domain modular structure for polysaccharide lyase family 4. FEBS Letters, 565, 188–194.

    Article  CAS  Google Scholar 

  39. Jensen, M. H., Otten, H., Christensen, U., Borchert, T. V., Christensen, L. L. H., Larsen, S., et al. (2010). Structural and biochemical studies elucidate the mechanism of rhamnogalacturonan lyase from Aspergillus aculeatus. Journal of Molecular Biology, 404, 100–111.

    Article  CAS  Google Scholar 

  40. Wong, D. (2008). Enzymatic deconstruction of backbone structures of the ramified regions in pectins. Protein Journal, 27, 30–42.

    Article  CAS  Google Scholar 

  41. Bonnin, E., Garnier, C., & Ralet, M. C. (2014). Pectin-modifying enzymes and pectin-derived materials: applications and impacts. Applied Microbiology and Biotechnology, 98, 519–532.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by JSPS KAKENHI Grant Number 25450135.

Author information

Authors and Affiliations

  1. Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka, 599-8531, Japan

    Marin Iwai, Hiroyuki Yamada, Takeshi Ikemoto, Shotaro Matsumoto & Tatsuji Sakamoto

  2. Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Sakai, Osaka, 599-8531, Japan

    Daisuke Fujiwara

  3. Division of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka, 598-8531, Japan

    Shigeo Takenaka

Authors
  1. Marin Iwai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hiroyuki Yamada
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Takeshi Ikemoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shotaro Matsumoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Daisuke Fujiwara
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Shigeo Takenaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tatsuji Sakamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tatsuji Sakamoto.

Additional information

M. Iwai and H. Yamada are equal first authors.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Iwai, M., Yamada, H., Ikemoto, T. et al. Biochemical Characterization and Overexpression of an Endo-rhamnogalacturonan Lyase from Penicillium chrysogenum . Mol Biotechnol 57, 539–548 (2015). https://doi.org/10.1007/s12033-015-9847-4

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-015-9847-4

Keywords

Search

Navigation