Skip to main content

Advertisement

SpringerLink
Log in

A New Subtilase-Like Protease Deriving from Fusarium equiseti with High Potential for Industrial Applications

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

A gene encoding a novel extracellular subtilisin-like protease was cloned from the ascomycete Fusarium equiseti and expressed in Trichoderma reesei. The F. equiseti protease (Fe protease) showed excellent performance in stain removal and good compatibility with several commercial laundry detergent formulations, suggesting that it has high potential for use in various industrial applications. The recombinant enzyme was purified and characterized. The temperature optimum of the Fe protease was 60 °C and it showed high activity in the pH range of 6–10, with a sharp decline in activity at pH above 10. The amino acid specificity of the Fe protease was studied using casein, cytochrome c, and ubiquitin as substrates. The Fe protease had broad substrate specificity: almost all amino acid residues were accepted at position P1, even though it showed some preference for cleavage at the C-terminal side of asparagine and histidine residues. The S4 subsite of Fe protease favors aspartic acid and threonine. The other well-characterized proteases from filamentous fungi, Proteinase K from Engyodontium album, Thermomycolin from Malbranchea sulfurea, and alkaline subtilisins from Bacillus species prefer hydrophobic amino acids in both the S1 and S4 subsites. Due to its different specificity compared to the members of the S8 family of clan SB of proteases, we consider that the Fe protease is a new protease. It does not belong to any previously defined IUBMB groups of proteases.

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
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  1. Craik, C. S., Page, M. J., & Madison, E. L. (2011). Proteases as therapeutics. The Biochemical Journal, 435(1), 1–16.

    Article  CAS  Google Scholar 

  2. Gupta, R., Beg, Q. K., & Lorenz, P. (2002). Bacterial alkaline proteases: molecular approaches and industrial applications. Applied Microbiology and Biotechnology, 59, 15–32.

    Article  CAS  Google Scholar 

  3. Singhal, P., Nigam, V. K., & Vidyarthi, A. S. (2012). Studies on production, characterization and applications of microbial alkaline proteases. International Journal of Advanced Biotechnology and Research, 3(3), 653–669.

    CAS  Google Scholar 

  4. Siezen, R. J., & Leunissen, J. A. (1997). Subtilases: the superfamily of subtilisin-like serine proteases. Protein Science, 6(3), 501–523.

    Article  CAS  Google Scholar 

  5. Tomkinson, B. & Eklund, S. (2013). Tripeptidyl-peptidase II. In N. D. Rawlings, G. Salvesen (Eds.), Handbook of Proteolytic Enzymes. Volume 3. 3rd (pp. 3325–3331). Elsevier.

  6. Maurer, K. H. (2004). Detergent proteases. Current Opinion in Biotechnology, 15(4), 330–334.

    Article  CAS  Google Scholar 

  7. Betzel, C., Pal, G. P., & Saenger, W. (1988). Three-dimensional structure of proteinase K at 0.15-nm resolution. European Journal of Biochemistry, 178(1), 155–171.

    Article  CAS  Google Scholar 

  8. Kraus, E., & Femfert, U. (1976). Proteinase K from the mold Tritirachium album Limber. Specificity and mode of action. Hoppe-Seyler’s Zeitschrift für physiologische Chemie, 357, 937–947.

    Article  CAS  Google Scholar 

  9. Lizardi, P. M., & Engelberg, A. (1979). Rapid isolation of RNA using proteinase K and sodium perchlorate. Analytical Biochemistry, 98(1), 116–122.

    Article  CAS  Google Scholar 

  10. Ebeling, W., Hennrich, N., Klockow, M., Metz, H., Orth, H. D., & Lang, H. (1974). Proteinase K from Tritirachium album Limber. European Journal of Biochemistry, 47, 91–97.

    Article  CAS  Google Scholar 

  11. Guo, J.-P., & Ma, Y. (2008). High-level expression, purification and characterization of recombinant Aspergillus oryzae alkaline protease in Pichia pastoris. Protein Expression and Purification, 58(2), 301–308.

    Article  CAS  Google Scholar 

  12. Ong, P. S., & Gaucher, G. M. (1976). Production, purification and characterization of thermomycolase, the extracellular serine protease of the thermophilic fungus Malbranchea pulchella var. sulfurea. Canadian Journal of Microbiology, 22(2), 165–176.

    Article  CAS  Google Scholar 

  13. Stevenson, K. J., & Gaucher, G. M. (1975). The substrate specificity of thermomycolase, an extracellular serine proteinase from the thermophilic fungus Malbranchea pulchella var. sulfurea. The Biochemical Journal, 151(3), 527–542.

    Article  CAS  Google Scholar 

  14. Liang, L., Meng, Z., Ye, F., Yang, J., Liu, S., Sun, Y., et al. (2010). The crystal structures of two cuticle-degrading proteases from nematophagous fungi and their contribution to infection against nematodes. FASEB Journal, 24(5), 1391–1400.

    Article  CAS  Google Scholar 

  15. Pähler, A., Banerjee, A., Dattagupta, J. K., Fujiwara, T., Lindner, K., Pal, G. P., et al. (1984). Three-dimensional structure of fungal proteinase K reveals similarity to bacterial subtilisin. The EMBO Journal, 3(6), 1311–1314.

    Google Scholar 

  16. Siezen, R. J., Renckens, B., & Boekhorst, J. (2007). Evolution of prokaryotic subtilases: genome-wide analysis reveals novel subfamilies with different catalytic residues. Proteins: Structure, Function, and Bioinformatics, 67(3), 681–694.

    Article  CAS  Google Scholar 

  17. Parry, D. W., Jenkinson, P., & McLeod, L. (1995). Fusarium ear blight (scab) in small grain cereals—a review. Plant Pathology, 44, 207–238.

    Article  Google Scholar 

  18. Castro, I. M., Lima, A. A., Paula, C. A., Nicoli, J. R., & Brandão, R. L. (1991). Effect of substrate and pH on the activity of proteases from Fusarium oxysporum var. lini. Journal of Fermentation and Bioengineering, 72(2), 132–134.

    Article  CAS  Google Scholar 

  19. Kumari, B. L., Vijetha, P., & Sudhakar, P. (2010). Optimization of physico-chemical properties for production of alkaline protease from Fusarium graminearum. Recent Research in Science and Technology, 2(4), 24–28.

    Google Scholar 

  20. Dunaevskii, Y. E., Matveeva, A. R., Fatkhullina, G. N., Belyakova, G. A., Kolomiets, T. M., Kovalenko, E. D., et al. (2008). Extracellular proteases of mycelial fungi as participants of pathogenic process. Russian Journal of Bioorganic Chemistry, 34(3), 286–289.

    Article  CAS  Google Scholar 

  21. Pekkarinen, A. I., & Jones, B. L. (2002). Trypsin-like proteinase produced by Fusarium culmorum grown on grain proteins. Journal of Agricultural and Food Chemistry, 50(13), 3849–3855.

    Article  CAS  Google Scholar 

  22. Choudhuri, S., DiNovi, M., Leblanc, J.-C., Meyland, I., Mueller, U., & Srinivasan, J. (2012). Serine protease (trypsin) from Fusarium oxysporum expressed in Fusarium venenatum. FAO JECFA Monographs, 13, 51–61.

    Google Scholar 

  23. Perona, J. J., & Craik, C. S. (1995). Structural basis of substrate specificity in the serine proteases. Protein Science, 4(3), 337–360.

    Article  CAS  Google Scholar 

  24. Olivieri, F., Zanetti, M. E., Oliva, C. R., Covarrubias, A. A., & Casalongue, C. A. (2002). Characterization of an extracellular serine protease of Fusarium eumartii and its action on pathogenesis related proteins. European Journal of Plant Pathology, 108, 63–72.

    Article  CAS  Google Scholar 

  25. Pekkarinen, A. I., Jones, B. L., & Niku-Paavola, M.-L. (2002). Purification and properties of an alkaline proteinase of Fusarium culmorum. European Journal of Biochemistry, 269, 798–807.

    Article  CAS  Google Scholar 

  26. DelMar, E. G., Largman, C., Brodrick, J. W., & Geokas, M. C. (1979). A sensitive new substrate for chymotrypsin. Analytical Biochemistry, 99(2), 316–320.

    Article  CAS  Google Scholar 

  27. Gasteiger, E., Gattiker, A., Hoogland, C., Ivanyl, I., Appel, R. D., & Balroch, A. (2003). ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Research, 31(13), 3784–3788.

    Article  CAS  Google Scholar 

  28. Shevchenko, A., Wilm, M., Vorm, O., Jensen, O. N., Podtelejnikov, A. V., Neubauer, G., et al. (1996). A strategy for identifying gel-separated proteins in sequence databases by MS alone. Biochemical Society Transactions, 24(3), 893–896.

    Article  CAS  Google Scholar 

  29. Poutanen, M., Salusjärvi, L., Ruohonen, L., Penttilä, M., & Kalkkinen, N. (2001). Use of matrix-assisted laser desorption/ionization time-of-flight mass mapping and nanospray liquid chromatography/electrospray ionization tandem mass spectrometry sequence tag analysis for high sensitivity identification of yeast proteins separated by two-dimensional gel electrophoresis. Rapid Communications in Mass Spectrometry, 15(18), 1685–1692.

    Article  CAS  Google Scholar 

  30. Sambrook, J., & Russell, D. W. (2001). Molecular cloning. A laboratory manual (3rd ed., pp. 1–3). Cold Spring Harbor: Cold Spring Harbor Laboratory Press (CSHL Press).

    Google Scholar 

  31. Raeder, U., & Broda, P. (1985). Rapid preparation of DNA from filamentous fungi. Letters in Applied Microbiology, 1(1), 17–20.

    Article  CAS  Google Scholar 

  32. Gurr, S. J., Unkles, S. E., & Kinghorn, J. R. (1987). The structure and organization of nuclear genes of filamentous fungi. In J. R. Kinghorn (Ed.), Gene structure in eukaryotic microbes (pp. 93–139). Washington: Special Publications of the Society for General Microbiology.

    Google Scholar 

  33. Bendtsen, J. D., Nielsen, H., von Heijne, G., & Brunak, S. (2004). Improved prediction of signal peptides: signalIP 3.0. Journal of Molecular Biology, 340(4), 783–795.

    Article  Google Scholar 

  34. Paloheimo, M., Mäntylä, A., Kallio, J., & Suominen, P. (2003). High-yield production of a bacterial xylanase in the filamentous fungus Trichoderma reesei requires a carrier polypeptide with an intact domain structure. Applied and Environmental Microbiology, 69(12), 7073–7082.

    Article  CAS  Google Scholar 

  35. Penttilä, M., Nevalainen, H., Rättö, M., Salminen, E., & Knowles, J. (1987). A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene, 61(2), 155–164.

    Article  Google Scholar 

  36. Karhunen, T., Mantyla, A., Nevalainen, K. M., & Suominen, P. L. (1993). High frequency one-step gene replacement in Trichoderma reesei. I. Endoglucanase I overproduction. Molecular and General Genetics MGG, 241(5–6), 515–522.

    Article  CAS  Google Scholar 

  37. Joutsjoki, V. V., Torkkeli, T. K., & Nevalainen, K. M. (1993). Transformation of Trichoderma reesei with the Hormoconis resinae glucoamylase P (gamP) gene: production of a heterologous glucoamylase by Trichoderma reesei. Current Genetics, 24(3), 223–228.

    Article  CAS  Google Scholar 

  38. Britton, H.T.S. & Robinson, R.A. (1931). CXCVIII: universal buffer solutions and the dissociation constant of veronal. Journal of the Chemical Society C: Organic, 1456–1462.

  39. Davies, M. T. (1959). A universal buffer solution for use in ultra-violet spectrophotometry. Analyst, 84, 248–251.

    Article  CAS  Google Scholar 

  40. Rawlings, N. D., Waller, M., Barrett, A. J., & Bateman, A. (2013). MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Research, 42(D1), D503.

    Article  Google Scholar 

  41. Georgieva, D., Genov, N., Voelter, W., & Betzel, C. (2006). Catalytic efficiencies of alkaline proteinases from microorganisms. Zeitschrift für Naturforschung. C, 61(5–6), 445–452.

    CAS  Google Scholar 

  42. Taguchi, S., Ozaki, A., & Momose, H. (1998). Engineering of a cold-adapted protease by sequential random mutagenesis and a screening system. Applied and Environmental Microbiology, 64(2), 492–495.

    CAS  Google Scholar 

  43. Tindbaek, N., Svendsen, A., Oestergaard, P. R., & Draborg, H. (2004). Engineering a substrate-specific cold-adapted subtilisin. Protein Engineering Design and Selection, 17(2), 149–156.

    Article  CAS  Google Scholar 

  44. Betzel, C., Klupsch, S., Branner, S., & Wilson, K. S. (1996). Crystal structures of the alkaline proteases savinase and esperase from Bacillus lentus. Advances in Experimental Medicine and Biology, 379, 49–61.

    Article  CAS  Google Scholar 

  45. Horn, J. R., Ramaswamy, S., & Murphy, K. P. (2003). Structure and energetics of protein–protein interactions: the role of conformational heterogeneity in OMTKY3 binding to serine proteases. Journal of Molecular Biology, 331(2), 497–508.

    Article  CAS  Google Scholar 

  46. Betzel, C., Gourinath, S., Kumar, P., Kaur, P., Perbandt, M., Eschenburg, S., et al. (2001). Structure of a serine protease Proteinase K from Tritirachium album limber at 0.98 Å resolution. Biochemistry, 40, 3080–3088.

    Article  CAS  Google Scholar 

  47. Savitha, S., Sadhasivam, S., Swaminathan, K., & Lin, F. H. (2011). Fungal protease: production, purification and compatibility with laundry detergents and their wash performance. Journal of the Taiwan Institute of Chemical Engineers, 42(2), 298–304.

    Article  CAS  Google Scholar 

  48. Cavello, I. A., Hours, R. A., & Cavalitto, S. F. (2012). Bioprocessing of “hair waste” by Paecilomyces lilacinus as a source of a bleach-stable, alkaline, and thermostable keratinase with potential application as a laundry detergent additive: characterization and wash performance analysis. Biotechnology Research International, 2012, 369308.

    Article  Google Scholar 

  49. Niyonzima, F. N., & More, S. (2014). Purification and properties of detergent-compatible extracellular alkaline protease from Scopulariopsis spp. Preparative Biochemistry & Biotechnology, 44(7), 738–759.

    Article  CAS  Google Scholar 

  50. Estell, D. A., Grayear, T. P., & Wells, J. A. (1985). Engineering an enzyme by site-directed mutagenesis to be resistant to chemical oxidation. The Journal of Biological Cehemistry, 260(11), 6518–6521.

    CAS  Google Scholar 

  51. Vojcic, L., Despotovic, D., Maurer, K.-H., Zacharias, M., Bocola, M., Martinez, R., et al. (2013). Reengineering of subtilisin Carlsberg for oxidative resistance. Biological Chemistry, 394(1), 79–87.

    Article  CAS  Google Scholar 

  52. Grøn, H., Bech, L. M., Branner, S., & Breddam, K. (1990). A highly active and oxidation-resistant subtilisin-like enzyme produced by a combination of site-directed mutagenesis and chemical modification. European Journal of Biochemistry, 194(3), 897–901.

    Article  Google Scholar 

Download references

Acknowledgments

The authors wish to thank Gunilla Rönnholm of the Institute of Biotechnology in Helsinki for her contribution on determination of the peptide sequences. The authors are grateful to Karl-Heinz Maurer for critically reviewing the manuscript.

Author information

Authors and Affiliations

  1. Roal Oy, Tykkimäentie 15, 05200, Rajamäki, Finland

    Kari Juntunen, Susanna Mäkinen & Marja Paloheimo

  2. Department of Chemistry, University of Eastern Finland, PO BOX 111, 80101, Joensuu, Finland

    Sari Isoniemi & Janne Jänis

  3. AB Enzymes Oy, Tykkimäentie 15, 05200, Rajamäki, Finland

    Leena Valtakari

  4. Institute of Molecular Enzyme Technology, Research Centre Juelich, Heinrich-Heine-University Duesseldorf, 52426, Juelich, Germany

    Alexander Pelzer

Authors
  1. Kari Juntunen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Susanna Mäkinen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sari Isoniemi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Leena Valtakari
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Alexander Pelzer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Janne Jänis
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Marja Paloheimo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kari Juntunen.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Juntunen, K., Mäkinen, S., Isoniemi, S. et al. A New Subtilase-Like Protease Deriving from Fusarium equiseti with High Potential for Industrial Applications. Appl Biochem Biotechnol 177, 407–430 (2015). https://doi.org/10.1007/s12010-015-1752-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-015-1752-6

Keywords

Search

Navigation