Skip to main content
SpringerLink
Log in

The Proteolytic Activity of Philibertia gilliesii Latex. Purification of Philibertain g II

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

Abstract

The latex from the patagonic plant Philibertia gilliesii Hook. et Arn. (Apocynaceae) is a milky-white suspension containing a proteolytic system constituted by several cysteine endopeptidases. A proteolytic preparation (philibertain g) from the latex of P. gilliesii fruits was obtained and characterized to evaluate its potential use in bioprocesses. Philibertain g contained 1.2 g/L protein and a specific (caseinolytic) activity of 7.0 Ucas/mg protein. It reached 80 % of its maximum caseinolytic activity in the pH 7–10 range, retained 80 % of the original activity after 2 h of incubation at temperatures ranging from 25 to 45 °C and could be fully inactivated after 5 min at 75 °C. Philibertain g retained 60 % of the initial activity even at 1 M NaCl and was able to hydrolyze proteins from stickwater one, of the main waste effluents generated during fishmeal production. Furthermore, as a contribution to the knowledge of the proteolytic system of P. gilliesii, we are reporting the purification of a new peptidase, named philibertain g II (pI 9.4, molecular mass 23,977 Da, N-terminus LPESVDWREKGVVFPXRNQ) isolated from philibertain g through a purification scheme including acetone fractionation, cation exchange, molecular exclusion chromatography, and ultrafiltration.

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

Similar content being viewed by others

References

  1. Feijoo-Siota, L., & Villa, T. G. (2011). Native and biotechnologically engineered plant proteases with industrial applications. Food Bioprocess Technol, 4, 1066–88.

    Article  CAS  Google Scholar 

  2. Pardo, M. F., López, L. M. I., Canals, F., Avilés, F. X., Natalucci, C. L., & Caffini, N. O. (2000). Purification of balansain I, an endopeptidase from unripe fruits of Bromelia balansae Mez (Bromeliaceae). J Agric Food Chem, 48, 3795–800.

    Article  CAS  Google Scholar 

  3. González-Rábade, N., Badillo-Corona, J. A., Aranda-Barradas, J. S., & Oliver-Salvador, M. D. C. (2011). Production of plant proteases in vivo and in vitro—a review. Biotechnol Adv, 29, 983–96.

    Article  Google Scholar 

  4. Yadav, R. E., Patel, A. K., & Jagannadham, M. V. (2011). Purification and biochemical characterization of a chymotrypsin like serine protease from Euphorbia neriifolia Linn. Process Biochem, 46, 1654–62.

    Article  CAS  Google Scholar 

  5. Lynn, K. R., Brockbank, W. J., & Clevette-Radford, N. A. (1980). Multiple forms of the asclepains, cisteinyl proteases from milkweed. Biochim Biophys Acta, 612, 119–25.

    Article  CAS  Google Scholar 

  6. Ramos, M. V., Araújo, E. S., Jucá, T. L., Monteiro-Moreira, A. C., Vasconcelos, I. M., Moreira, R. A., et al. (2013). New insights into the complex mixture of latex cysteine peptidases in Calotropis procera. Int J Biol Macromol, 58, 211–9.

    Article  CAS  Google Scholar 

  7. Ramos, M. V., Souza, D. P., Gomes, M. T., Freitas, C. D., Carvalho, C. P., Júnior, P. A., et al. (2014). A phytopathogenic cysteine peptidase from latex of wild rubber vine Cryptostegia grandiflora. Protein J, 33, 199–209.

    Article  CAS  Google Scholar 

  8. Pal, G., & Sinha, N. K. (1980). Isolation, crystallization, and properties of calotropins DI and DII from Calotropis gigantea. Arch Biochem Biophys, 202, 321–9.

    Article  CAS  Google Scholar 

  9. Nallamsetty, S., Kundu, S., & Jagannadham, M. V. (2003). Purification and biochemical characterization of a highly active cysteine protease ervatamin a from the latex of Ervatamia coronaria. J Protein Chem, 22, 1–13.

    Article  CAS  Google Scholar 

  10. Patel, B. K., & Jagannadham, M. V. (2003). A high cysteine containing thiol proteinase from the latex of Ervatamia heyneana: purification and comparison with ervatamin B and C from Ervatamia coronaria. J Agric Food Chem, 51, 6326–34.

    Article  CAS  Google Scholar 

  11. Tomar, R., Kumar, R., & Jagannadham, M. V. (2008). A stable serine protease, wrightin, from the latex of the plant Wrightia tinctoria (Roxb.) R.Br.: purification and biochemical properties. J Agric Food Chem, 56, 1479–87.

    Article  CAS  Google Scholar 

  12. Singh, A. N., Shukla, A. K., Jagannadham, M. V., & Dubey, V. K. (2010). Purification of a novel cysteine protease, Procerain B, from Calotropis procera with distinct characteristics compared to Procerain. Process Biochem, 45, 399–406.

    Article  CAS  Google Scholar 

  13. Singh, A. N., & Dubey, V. K. (2011). Exploring applications of procerain b, a novel protease from Calotropis procera, and characterization by N-terminal sequencing as well as peptide mass fingerprinting. Appl Biochem Biotechnol, 164, 573–80.

    Article  CAS  Google Scholar 

  14. Priolo, N., Morcelle del Valle, S., Arribére, M. C., López, L. M. I., & Caffini, N. O. (2000). Isolation and characterization of a cysteine protease from the latex of Araujia hortorum fruits. J Protein Chem, 19, 39–48.

    Article  CAS  Google Scholar 

  15. Obregón, W. D., Arribére, M. C., Morcelle del Valle, S., Liggieri, C., Caffini, N. O., & Priolo, N. S. (2001). Two new cysteine endopeptidases obtained from the latex of Araujia hortorum fruits. J Protein Chem, 20, 317–25.

    Article  Google Scholar 

  16. Cavalli, S. V., Cortadi, A., Arribére, M. C., Conforti, P., Caffini, N. O., & Priolo, N. S. (2001). Comparison of two cysteine endopeptidases from latices of Morrenia brachystephana Griseb. and Morrenia odorata (Hook et Arn.) Lindley (Asclepiadaceae). Biol Chem Hoppe Seyler, 382, 879–83.

    CAS  Google Scholar 

  17. Trejo, S. A., López, L. M. I., Cimino, C. V., Caffini, N. O., & Natalucci, C. L. (2001). Purification and characterization of a new plant endopeptidase isolated from the latex of Asclepias fruticosa L. (Asclepiadaceae). J Protein Chem, 20, 469–77.

    Article  CAS  Google Scholar 

  18. Vairo Cavalli, S. E., Arribére, M. C., Cortadi, A., Caffini, N. O., & Priolo, N. S. (2003). Morrenain b I, a papain-like endopeptidase from the latex of Morrenia brachystephana Griseb. (Asclepiadaceae). J Protein Chem, 22, 15–22.

    Article  CAS  Google Scholar 

  19. Morcelle, S. R., Trejo, S. A., Canals, F., Aviles, F. X., & Priolo, N. S. (2004). Funastrain cII: a cysteine endopeptidase purified from the latex of Funastrum clausum. Protein J, 23, 205–15.

    Article  CAS  Google Scholar 

  20. Liggieri, C. S., Arribére, M. C., Trejo, S. A., Canals, F., Aviles, F. X., & Priolo, N. S. (2004). Purification and biochemical characterization of asclepain cI from the latex of Asclepias curassavica L. Protein J, 23, 403–11.

    Article  CAS  Google Scholar 

  21. Sequeiros, C., Torres, M. J., Trejo, S. A., Esteves, J. L., Natalucci, C. L., & López, L. M. I. (2005). Philibertain g I, the most basic cysteine endopeptidase purified from the latex of Philibertia gilliesii Hook. et Arn. (Apocynaceae). Protein J, 24, 445–53.

    Article  CAS  Google Scholar 

  22. Liggieri, C., Obregón, W., Trejo, S., & Priolo, N. (2009). Biochemical analysis of a papain-like protease isolated from the latex of Asclepias curassavica L. Acta Biochim Biophys Sin, 41, 154–62.

    Article  CAS  Google Scholar 

  23. Trejo, S. A., López, L. M., Caffini, N. O., Natalucci, C. L., Canals, F., & Avilés, F. X. (2009). Sequencing and characterization of asclepain f: the first cysteine peptidase cDNA cloned and expressed from Asclepias fruticosa latex. Planta, 230, 319–28.

    Article  CAS  Google Scholar 

  24. Correa, M. N. (1999). Parte VI. Dicotiledóneas. In F. Patagónica (Ed.), Gamopétalas (Ericaceae a Calyceraceae) (p. 536). Buenos Aires: Colección Científica del INTA.

    Google Scholar 

  25. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of micrograms quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 72, 248–54.

    Article  CAS  Google Scholar 

  26. López, L. M. I., Sequeiros, C., Natalucci, C. L., Brullo, A., Maras, B., Barra, D., & Caffini, N. O. (2000). Purification and characterization of macrodontain I, a cysteine peptidase from unripe fruits of Pseudananas macrodontes (Morr.) Harms (Bromeliaceae). Protein Expr Purif, 18, 133–40.

    Article  Google Scholar 

  27. Good, N. E., & Izawa, S. (1972). Hydrogen ion buffers. Methods Enzymol, 24, 53–68.

    Article  CAS  Google Scholar 

  28. del Valle, J. M., & Aguilera, J. M. (1991). Physicochemical characterisation of raw fish and stickwater from fish meal production. J Sci Food Agric, 54, 429–41.

    Article  Google Scholar 

  29. Schägger, H., & von Jagow, G. (1987). Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem, 166, 368–79.

    Article  Google Scholar 

  30. Westergaar, J. L., Hackbarth, C., Treuhaft, M. W., & Roberts, R. C. (1980). Detection of proteinases in electrophoretograms of complex mixtures. J Immunol Methods, 34, 167–75.

    Article  Google Scholar 

  31. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–5.

    Article  CAS  Google Scholar 

  32. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., et al. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res, 25, 3389–402.

    Article  CAS  Google Scholar 

  33. Bindhu, O. S., & Singh, M. K. (2014). Hemostatic, milk clotting and blood stain removal potential of cysteine proteases from Calotropis gigantea (L.) R. Br. Latex. Pharmacogn Mag, 10, S350–6.

    Article  Google Scholar 

  34. Rajesh, R., Raghavendra Gowda, C. D., Nataraju, A., Dhananjaya, B. L., Kemparaju, K., & Vishwanath, B. S. (2005). Procoagulant activity of Calotropis gigantea latex associated with fibrin(ogen)olytic activity. Toxicon, 46, 84–92.

    Article  CAS  Google Scholar 

  35. Ramos, M. V., Viana, C. A., Silva, A. F. B., Freitas, C. D. T., Figueiredo, I. S. T., Oliveira, R. S. B., et al. (2012). Proteins derived from latex of C. procera maintain coagulation homeostasis in septic mice and exhibit thrombin- and plasmin-like activities. Naunyn Schmiedebergs Arch Pharmacol, 385, 455–63.

    Article  CAS  Google Scholar 

  36. Biazus, J. P. M., Santana, J. C. C., & De Souza, R. R. (2006). Empirical modeling of biodegradation process of proteic effluents by enzymes of Carica papaya sp. Revista Brasileira de Engenharia Agrícola e Ambiental, 10, 436–40.

    Article  Google Scholar 

  37. Pitpreecha, S., & Damrongsakkul, S. (2006). Hydrolysis of raw hide using proteolytic enzyme extracted from papaya latex. Korean J Chem Eng, 23, 972–6.

    Article  CAS  Google Scholar 

  38. Torres, M. J., Trejo, S. A., Martin, M. I., Natalucci, C. L., Aviles, F. X., & Lopez, L. M. I. (2010). Purification and characterization of a cysteine endopeptidase from Vasconcellea quercifolia A. St.- Hil. latex displaying high substrate specificity. J Agric Food Chem, 58, 11027–35.

    Article  CAS  Google Scholar 

  39. Scopes, R. K. (1984). Protein purification. Principles and practice (pp. 219). New York: Springer.

    Google Scholar 

  40. Uhlig, H. (1998). Industrial enzymes and their applications (pp. 351–374). New York: Wiley-Interscience.

    Google Scholar 

  41. Glazer, A. N., & Smith, E. L. (1971). Papain and other plant sulfhydryl proteolytic enzymes. In P. D. Boyer (Ed.), The Enzymes (pp. 501–46). New York: Academic.

    Google Scholar 

  42. Sundd, M., Kundu, S., Pal, G. P., & Jagannadham, M. V. (1998). Purification and characterization of a highly stable cysteine peptidase from the latex of Ervatamia coronaria. Biosci Biotechnol Biochem, 62, 1947–55.

    Article  CAS  Google Scholar 

  43. Kundu, S., Sundd, M., & Jagannadham, M. V. (2000). Purification and characterization of a stable cysteine protease Ervatamin B, with two disulfide bridges, from latex of Ervatamia coronaria. J Agric Food Chem, 48, 171–9.

    Article  CAS  Google Scholar 

  44. Dubey, V. K., & Jagannadham, M. V. (2003). Procerain, a stable cysteine peptidase from the latex of Calotropis procera. Phytochemistry, 62, 1057–71.

    Article  Google Scholar 

  45. Ahmad, B., & Khan, R. H. (2006). Studies on the acid unfolded and molten globule states of catalytically active stem bromelain: a comparison with catalytically inactive form. J Biochem, 140, 501–8.

    Article  CAS  Google Scholar 

  46. Bechtel, P. J. (2005). Properties of stickwater from fish processing byproducts. J Aquat Food Prod Technol, 14, 25–38.

    Article  CAS  Google Scholar 

  47. Domsalla, A., & Melzig, M. F. (2008). Occurrence and properties of proteases in plant lattices. Planta Med, 74, 699–711.

    Article  CAS  Google Scholar 

  48. Biswas, S., Chakrabarti, C., Kundu, S., Jagannadham, M. V., & Dattagupta, J. K. (2003). Proposed amino acid sequence and the 1.63 A X-ray crystal structure of a plant cysteine protease, ervatamin B: some insights into the structural basis of its stability and substrate specificity. Proteins, 51, 489–97.

    Article  CAS  Google Scholar 

  49. Thakurta, P. G., Biswas, S., Chakrabarti, C., Sundd, M., Jagannadham, M. V., & Dattagupta, J. K. (2004). Structural basis of the unusual stability and substrate specificity of ervatamin C, a plant cysteine protease from Ervatamia coronaria. Biochemistry, 43, 1532–40.

    Article  CAS  Google Scholar 

  50. Singh, A. N., Yadav, P., & Dubey, V. K. (2013). cDNA cloning and molecular modeling of procerain B, a novel cysteine endopeptidase isolated from Calotropis procera. PLoS ONE, 8, e59806.

    Article  CAS  Google Scholar 

  51. Brockbank, W. J., & Kenneth, R. L. (1979). Purification and preliminary characterization of two asclepains from the latex of Asclepias syriaca L. (Milkweed). Biochim Biophys Acta, 578, 13–22.

    Article  CAS  Google Scholar 

  52. Mitchel, R. E., Chaiken, R. M., & Smith, E. L. (1970). The complete amino acid sequence of papain. J Biol Chem, 245, 3485–92.

    CAS  Google Scholar 

  53. Rawlings, N. M., & Barrett, A. J. (2013). Introduction: the clans and families of cysteine peptidases. In N. D. Rawlings & G. S. Salvesen (Eds.), Handbook of proteolytic enzymes (pp. 1743–73). London: Academic.

    Chapter  Google Scholar 

Download references

Acknowledgments

The authors acknowledge Dr. Ana María Beeskow (CENPAT-CONICET) for the identification of plant material. L.M.I. López, M.J. Torres, and C. Sequeiros are members of the CONICET Researcher Career; C.L. Natalucci is member of the CICPBA Researcher Career. The present work was supported by grants from CONICET (PIP 0297), CICPBA, and University of La Plata (X-576), Argentina.

Author information

Authors and Affiliations

  1. CENPAT-CONICET, Centro Nacional Patagónico, Blvd. Brown N° 2915, 9120, Puerto Madryn, Chubut, Argentina

    Cynthia Sequeiros & Marina L. Nievas

  2. Centro de Investigaciones y Transferencia del Noroeste de la provincia de Buenos Aires, UNNOBA-CONICET, Junín, Argentina

    María J. Torres

  3. CIPROVE, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, CC 711, 1900, La Plata, Argentina

    Néstor O. Caffini & Claudia L. Natalucci

  4. CITEC, Instituto Nacional de Tecnología Industrial, Ciencias de la Salud - Universidad Nacional Arturo Jauretche, Buenos Aires, Argentina

    Laura M. I. López

  5. Servei de Proteòmicai Biologia Estructural (SePBioEs), Universitat Autònoma de Barcelona, Campus Universitari, 08193 Bellaterra, Cerdanyola del Vallès, Barcelona, Spain

    Sebastián A. Trejo

Authors
  1. Cynthia Sequeiros
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. María J. Torres
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marina L. Nievas
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Néstor O. Caffini
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Claudia L. Natalucci
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Laura M. I. López
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sebastián A. Trejo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Laura M. I. López.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sequeiros, C., Torres, M.J., Nievas, M.L. et al. The Proteolytic Activity of Philibertia gilliesii Latex. Purification of Philibertain g II. Appl Biochem Biotechnol 179, 332–346 (2016). https://doi.org/10.1007/s12010-016-1997-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-016-1997-8

Keywords

Search

Navigation