Advertisement

Applied Biochemistry and Biotechnology

, Volume 182, Issue 2, pp 831–845 | Cite as

Purification and Biochemical Characterization of a Neutral Serine Protease from Trichoderma harzianum. Use in Antibacterial Peptide Production from a Fish By-Product Hydrolysate

  • Neyssene AissaouiEmail author
  • Jean-Marc Chobert
  • Thomas Haertlé
  • M. Nejib Marzouki
  • Ferid Abidi
Article

Abstract

This study reports the purification and biochemical characterization of an extracellular neutral protease from the fungus Trichoderma harzianum. The protease (Th-Protease) was purified from the culture supernatant to homogeneity by a three-step procedure with 14.2% recovery and 9.06-fold increase in specific activity. The purified enzyme appeared as a single protein band after sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of about 20 kDa. The optimum pH and temperature for the proteolytic activity were pH 7.0 and 40 °C, respectively. The enzyme was then investigated for its potential application in the production of antibacterial peptides. Interestingly, Scorpaena notata viscera protein hydrolysate prepared using the purified serine protease (Th-Protease) showed remarkable in vitro antibacterial activities. A peptide with a high antibacterial activity was further purified by a three-step procedure, and its sequence was identified as FPIGMGHGSRPA. The result of this study offers a promising alternative to produce natural antibacterial peptides from fish protein hydrolysate.

Keywords

Trichoderma harzianum Protease Purification Protein hydrolysate Antibacterial peptide 

Notes

Acknowledgements

This work was supported by the financial project of LIP-MB Laboratory, INSAT, Carthage University, and the Ministry of Higher Education and Scientific Research of Tunisia.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Vishwanatha, K. S., Appu Rao, A. G., & Singh, S. A. (2009). Characterisation of acid protease expressed from Aspergillus oryzae MTCC 5341. Food Chemistry, 114, 402–407.CrossRefGoogle Scholar
  2. 2.
    Rao, M. B., Tanksale, A. M., Ghatge, M. S., & Deshpande, V. V. (1998). Molecular and biotechnological aspects of microbial proteases. Microbiology and Molecular Biology Reviews, 62, 597–635.Google Scholar
  3. 3.
    Zanphorlin, L. M., Cabral, H., Arantes, E., Assis, D., Juliano, L., Juliano, M. A., Da-Silva, R., Gomes, E., & Bonilla-Rodriguez, G. O. (2011). Purification and characterization of a new alkaline serine protease from the thermophilic fungus Myceliophthora sp. Process Biochemistry, 46, 2137–2143.CrossRefGoogle Scholar
  4. 4.
    Archer, D. B., & Peberdy, J. F. (1997). The molecular biology of secreted enzyme production by fungi. Critical Reviews in Biotechnology, 17, 273–306.CrossRefGoogle Scholar
  5. 5.
    Devi, K., Rasheedha-Banu, M. A., Gnanaprabhal, G. R., Pradeep, B. V., & Palaniswamy, M. (2008). Purification, characterization of alkaline protease enzyme from native isolate of Aspergillus niger and its compatibility with commercial detergents. Indian Journal of Science and Technology, 1, 1–6.Google Scholar
  6. 6.
    Archer, D. B. (2000). Filamentous fungi as microbial cell factories for food use. Current Opinion in Biotechnology, 11, 478–483.CrossRefGoogle Scholar
  7. 7.
    Sathivel, S., Bechtel, P., Babbitt, J., Smiley, S., Crapro, C., & Reppond, K. (2003). Biochemical and functional properties of herring (Clupea harengus) by product hydrolysates. Journal of Food Science, 68, 2196–2200.CrossRefGoogle Scholar
  8. 8.
    FAO (2014). The state of world fisheries and aquaculture 2012: opportunities and challenges (pp. 4–7). Rome: Food and Agricultural Organization of the United Nation.Google Scholar
  9. 9.
    Kristinsson, H. G. (2007) In F. Shahidi (Ed.), Aquatic food protein hydrolysates: maximising the value of marine by-products (pp. 229–248). Wood head Publishing Ltd..Google Scholar
  10. 10.
    Kristinsson, H. G., & Rasco, B. A. (2000). Biochemical and functional properties of Atlantic salmon (Salmo salar) muscle hydrolyzed with various alkaline proteases. Journal of Agricultural and Food Chemistry, 48, 657–666.CrossRefGoogle Scholar
  11. 11.
    Thiansilakul, Y., Benjakul, S., & Shahidi, F. (2007). Compositions, functional properties and antioxidative activity of protein hydrolysates prepared from round scad (Decapterus maruadsi). Food Chemistry, 103, 1385–1394.CrossRefGoogle Scholar
  12. 12.
    Song, R., Wei, R. B., Luo, H. Y., & Wang, D. F. (2012). Isolation and characterization of an antibacterial peptide fraction from the pepsin hydrolysate of half-fin anchovy (Setipinna taty). Molecules, 17, 2980–2991.CrossRefGoogle Scholar
  13. 13.
    Bolscher, J. G. M., Van der Kraan, M. I. A., Nazmi, K., Kalay, H., Grün, C. H., Van’t Hof, W., Veerman, E. C. I., & Nieuw Amerongen, A. V. (2006). A one-enzyme strategy to release an antimicrobial peptide from the LFampin-domain of bovine lactoferrin. Peptides, 27, 1–9.CrossRefGoogle Scholar
  14. 14.
    Abidi, F., Aissaoui, N., Gaudin, J. C., Chobert, J. M., Haertlé, T., & Marzouki, M. N. (2013). MS analysis and molecular characterization of Botrytis cinerea protease Prot-2. Use in bioactive peptides production. Applied Biochemistry and Biotechnology, 170, 231–247.CrossRefGoogle Scholar
  15. 15.
    Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.CrossRefGoogle Scholar
  16. 16.
    Phillips, P. K., Prior, D., & Awes, B. D. (1984). A modified azoalbumin technique for the assay of proteolytic enzymes for use in blood group serology. Journal of Clinical Pathology, 3, 329–331.CrossRefGoogle Scholar
  17. 17.
    Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of head of bacteriophage T4. Nature, 257, 680–685.CrossRefGoogle Scholar
  18. 18.
    Adler-Nissen, J. (1986). In J. Adler-Nissen (Ed.), Enzymic hydrolysis of food proteins: a review of food hydrolysis specific areas (pp. 57–109). Copenhagen: Elsevier Applied Science Publishers.Google Scholar
  19. 19.
    Fattouch, S., Caboni, P., Coroneo, V., Tuberoso, C. I. G., Angioni, A., & Dessi, S. (2007). Antimicrobial activity of Tunisian quince (Cydonia oblonga Miller) pulp and peel polyphenolic extracts. Journal of Agricultural and Food Chemistry, 55, 963–969.CrossRefGoogle Scholar
  20. 20.
    Gaida, A. V., Rudenskaia, G. N., & Stepanov, V. M. (1981). Isolation and comparative properties of serine proteinases of the microscopic fungi Trichoderma lignorum and Trichoderma koningii. Biochimie, 46, 2064–2073.Google Scholar
  21. 21.
    De Marco, J. L., & Felix, C. R. (2002) Characterization of a protease produced by a Trichoderma harzianum isolate which controls cocoa plant witches’ broom disease. BMC Biochemistry, 3, http://www.biomedcentral.com/1471-2091/3/3.
  22. 22.
    Singh, A., Srivastava, S., & Singh, H. B. (2008). Effect of substrates on growth and shelflife of Trichoderma harzianum and its use in biocontrol of disease. Bioresource Technology, 92, 470–473.Google Scholar
  23. 23.
    Jayalakshmi, S. K., Raju, S., Usha, S. R., Benagi, V. I., & Sreeramulu, V. I. (2009). Trichoderma harzianum L1 as a potential source for lytic enzymes and elicitor of defense responses in chickpea (Cicer arietinum L.) against wilt disease caused by Fusarium oxysporum f. sp. ciceri. Australian Journal of Crop Science, 3, 44–52.Google Scholar
  24. 24.
    Uchikoba, T., Mase, T., Arima, K., Yonezawa, H., & Kaneda, M. (2001). Isolation and characterization of a trypsin-like protease from Trichoderma viride. Biological Chemistry, 382, 1509–1513.CrossRefGoogle Scholar
  25. 25.
    Manonmani, H. K., & Joseph, R. (1993). Purification and properties of an extracellular proteinase of Trichoderma koningii. Enzyme and Microbial Technology, 15, 624–628.CrossRefGoogle Scholar
  26. 26.
    Eneyskaya, E. V., Kulminskaya, A. A., Savelev, A. N., Saveleva, N. V., Shabalin, K. A., & Neustroev, K. N. (1999). Acid protease from Trichoderma reesei: limited proteolysis of fungal carbohydrases. Applied Microbiology and Biotechnology, 52, 226–231.CrossRefGoogle Scholar
  27. 27.
    Dunaevsky, Y. E., Gruban, T. N., Beliakova, G. A., & Belozersky, M. A. (2000). Enzymes secreted by filamentous fungi: regulation of secretion and purification of an extracellular protease of Trichoderma harzianum. Biochemistry (Moscow), 65, 723–727.Google Scholar
  28. 28.
    Suarez, B., Rey, M., Castillo, P., Monte, E., & Llobell, A. (2004). Isolation and characterization of PRA1, a trypsin-like protease from the biocontrol agent Trichoderma harzianum CECT 2413 displaying nematicidal activity. Applied Microbiology and Biotechnology, 65, 46–55.CrossRefGoogle Scholar
  29. 29.
    Klompong, V., Benjakul, S., Kantachote, D., & Shahidi, F. (2007). Antioxidative activity and functional properties of protein hydrolysate of yellow stripe trevally (Selaroides leptolepis) as influenced by the degree of hydrolysis and enzyme type. Food Chemistry, 102, 1317–1327.CrossRefGoogle Scholar
  30. 30.
    Aissaoui, N., Abidi, F., Mahat, S., & Marzouki, M. N. (2014). Purification and biochemical characterization of a novel protease from Penicillium digitatum—use in bioactive peptides production. Journal of Basic Microbiology, 54, 1–12.CrossRefGoogle Scholar
  31. 31.
    Salampessy, J., Phillips, M., Seneweera, S., & Kailasapathy, K. (2010). Release of antimicrobial peptides through bromelain hydrolysis of leather jacket (Meuchenia sp.) insoluble proteins. Food Chemistry, 120, 556–560.CrossRefGoogle Scholar
  32. 32.
    Valgas, C., Machado de Souza, M., Smânia, E. F. A., & Smânia Jr., A. (2007). Screening methods to determine antibacterial activity of natural products. Brazilian Journal of Microbiology, 38, 369–380.CrossRefGoogle Scholar
  33. 33.
    Bulet, P., Stocklin, R., & Menin, L. (2004). Anti-microbial peptides: from invertebrates to vertebrates. Immunological Reviews, 198, 169–184.CrossRefGoogle Scholar
  34. 34.
    Dong, X. Z., Xu, H. B., Huang, K. X., Liou, Q., & Zhou, J. (2002). The preparation and characterization of an antimicrobial polypeptide from the loach, Misgurnus anguillicaudatus. Protein Expression and Purification, 26, 235–242.CrossRefGoogle Scholar
  35. 35.
    Kim, S. S., Shim, M. S., Chung, J., Lim, D. Y., & Lee, B. J. (2007). Purification and characterization of antimicrobial peptides from the skin secretion of Rana dybowskii. Peptides, 28, 1532–1539.CrossRefGoogle Scholar
  36. 36.
    Asoodeh, A., Zardini, H. Z., & Chamani, J. (2011). Identification and characterization of two novel antimicrobial peptides, temporin-Ra and temporin-Rb, from skin secretions of the marsh frog (Rana ridibunda). Journal of Peptide Science, 18, 10–16.CrossRefGoogle Scholar
  37. 37.
    Chen, W., Yang, X., Yang, X., Zhai, L., Lu, Z., Liu, J., & Yu, H. (2008). Antimicrobial peptides from the venoms of Vespa bicolor Fabricius. Peptides, 29, 1887–1892.CrossRefGoogle Scholar
  38. 38.
    Yang, X., Hu, Y., Xu, S., Hu, Y., Meng, H., Guo, C., Liu, Y., Liu, J., Yu, Z., & Wang, H. (2013). Identification of multiple antimicrobial peptides from the skin of fine-spined frog, Hylarana spinulosa (Ranidae). Biochimie, 95, 2429–2436.CrossRefGoogle Scholar
  39. 39.
    Rajanbabu, V., & Chen, J. Y. (2011). Applications of antimicrobial peptides from fish and perspectives for the future. Peptides, 32, 415–420.CrossRefGoogle Scholar
  40. 40.
    Friedrich, C. L., Moyles, D., Beveridge, T. J., & Hancock, R. E. W. (2000). Antibacterial action of structurally diverse cationic peptides on gram-positive bacteria. Antimicrobial Agents and Chemotherapy, 44, 2086–2092.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Neyssene Aissaoui
    • 1
    Email author
  • Jean-Marc Chobert
    • 2
  • Thomas Haertlé
    • 2
  • M. Nejib Marzouki
    • 1
  • Ferid Abidi
    • 1
    • 2
  1. 1.Laboratory of Protein Engineering and Bioactive Molecules (LIP-MB), National Institute of Applied Sciences and TechnologyUniversity of CarthageTunis CedexTunisia
  2. 2.UR 1268, Biopolymères Interactions Assemblages, Equipe Fonctions et Interactions des ProtéinesINRANantes Cedex 3France

Personalised recommendations