Extraction purification and characterization of trypsin inhibitors from Andean seeds

  • Patricio Castillo Escuela Politécnica Nacional
  • Lorena Quinchuela Escuela Politécnica Nacional
  • Paulina Echeverría Escuela Politécnica Nacional
  • Gonzalo Jácome Escuela Politécnica Nacional
Keywords: Trypsin inhibitor, covalent immobilization, Amaranthus hybridus L

Abstract

This work established the conditions of covalent immobilization of trypsin on a Sepharose matrix, which could be applied for the purification of trypsin inhibitors. The higher values of retention of enzymatic activity and immobilized enzymatic activity were obtained with a Sepharose 6B-CL matrix, at room temperature, a pH value of 10.5, an enzymatic load of 25 mg/mL, and a minimum immobilization time of 12 hours, in order to obtain a stable immobilization. The most active trypsin inhibitors were selected through the comparison of, extracts obtained from the seeds of amaranth (Amaranthus caudatus L.), pea (Pisum sativum), lupine or “chocho” (Lupinus mutabilis), bean (Phaseolus vulgaris) and “sangorache” (Amaranthus hybridus L.). The inhibitors were partially purified using centrifugal ultrafiltration, heat treatment, and TCA precipitation. The permeated and retained fractions of “sangorache” were selected as the most active trypsin inhibitors, and they were selectively purified using affinity chromatography in a Trypsin - Glyoxyl - Sepharose 6B-CL matrix. The kinetic characterization showed the presence of two inhibitors; the first one corresponded to a competitive inhibitor, while the second one behaved as a mixed inhibitor.

Downloads

Download data is not yet available.

References

Aitken, A. y Laermonth, M. (2002). Protein determination by UV absorption. En Walker, J. (Ed.). Protocols Handbook (pp. 3-6). Totowa, Estados Unidos: Humana Press Inc
American Psychological Association. (2012). Publication manual of the American Psychological Association. Washington, DC: American Psychological Assoc.
Betancur, D., Guerrero, L., Hernández, V. y Marrufo, D. (2012). Inhibición de la Enzima Convertidora de Angiotensina I con hidrolizados proteicos de Jatropha curcas. Bioquímica clínica latinoamericana, 46(3), 385 – 393. Obtenido de http://www.scielo.org.ar/pdf/abcl/v46n3/v46n3a06.pdf .
Bonzón, E. (1996). Obtención de una Matriz de Afinidad de Tripsina-Glioxil-Sepharosa CL-4B para la Purificación del Inhibidor de Proteasas de Stichodactyla heliantus. (Trabajo de diploma). Universidad de la Habana, La Habana, Cuba.
Castillo, P., Gómez, C., Sinche, M., Duchicela, J., & Muñoz, R. (2012). Identificación de la actividad proteolítica en especies vegetales presentes en las provincias de Loja y Pastaza. Revista Politécnica, 31.
Chávez, M., González, Y., Hernández, A., Pascual, I., Reytor, M. y Rivero, M. (2011). Screening of protease inhibitory activity in extracts of five Ascidian species from Cuban coasts. Biotecnología Aplicada, 28(2), 77 – 82. Obtenido de http://scielo.sld.cu/pdf/bta/v28n2/bta 02211.pdf
Da Silva, A. M., Tavares, A. P., Rocha, C. M., Cristóvão, R. O., Teixeira, J. A., & Macedo, E. A. (2012). Immobilization of commercial laccase on spent grain. Process biochemistry, 47(7), 1095-1101. Obtenido de http://www.sciencedirect.com/science/article/pii/S1359511312001 353
Del Monte, A., Cutiño, B., González, J., Chávez, M., Díaz, J. (2014). Diseño racional de la inmovilización de proteínas: aplicaciones en cromatografía de afinidad y bioconversión enzimática. Anales de la Academia de Ciencias de Cuba. Obtenido de http://www.revistaccuba.cu/index.php/acc/article/viewFile/187/145
Dos Santos, E., Oliveira, A., Rabêlo, L., Uchôa, A. y Araujo, A. (2012). Affinity Chromatography as a key tool purify protein protease inhibitors from plants. Obtenido de http://www.Intech open.com/books/affinity-chromatography/affinity-chromatography-asa-key-tool-to-purify-protease-inhibitors-from-plants
Earlanger, B., Kokowski, N. y Cohen, W. (1961). The preparation and properties of two new chromogenic substrates of trypsin. Archives of Biochemistry and Biophysics, 95(2), 271-278.
Elizalde, A., Porilla, Y. y Chaparro, D. (2009). Factores antinutricionales en semillas. Facultad de Ciencias Agropecuarias, 7(1), 46. Obtenido de http://www.scielo.org.co/pdf/bsaa/v7n1/ v7n1a07.pdf
Freije, J., Mulder,P., Werkman, W., Rieux, L., Niederlander, H., Verpoorte, E., y Bischoff, R. (2005). Chemically modified, immobilized trypsin reactor with improved digestion efficiency. Journal of proteome research, 4(5), 1805-1813.
García, R., Salas, E., Del monte, A., Del Rivero, A., Guerra, Y. y Chávez M. (2009). Micro y nano-biotecnologías en la detección y caracterización de inhibidores de proteasas de interés biomédico. Revista Cubana de Física. 26(1). 76. Obtenido de http://www.fisica.uh.cu/ biblioteca/revcubfi/2009/vol.26-No.1/RCF-26-1-2009-76.pdf
Habib, M. y Majid, K. (2007). Plant protease inhibitors: a defense strategy in plants. Biotechnology and Molecular Biology, 2(3), 68-85.
Koolman, J. y Röhm, K. (2004). Bioquímica texto y atlas. Madrid, España: Editorial Médida Panamericana.
Li, Y., Gao, F., Wei, W., Qu, J., Ma, G. y Zhou, W. (2010). Pore size of macroporous polystyrene microspheres affects lipase immobilization. Journal of Molecular Catalysis, 66 (1), 182-189.
Montgomery, D. (2003). Diseño y análisis de experimentos. México D.F., México: Limusa.
Müller-Esterl, F. (2008). Bioquímica. Fundamentos para medicina y ciencias de la vida. Obtenido de: http://books.google.com.ec/books?id=X2YV G6Fzp1UC&printsec=frontcover#v=one page&q&f=false
Nelson D. y Cox, M. (2014). Principles of biochemistry. Obtenido de https://archive.org/details/9788582710739
Ramírez, J., y Rangel, I. (2011). El frijol (Phaseolus vulgaris): su importancia nutricional y como fuente de fitoquímicos. Revista Fuente, 3(8).
Rocha, C., Gonçalves, P., y Teixeira, J. (2011). Immobilization of trypsin on spent grains for whey protein hydrolysis. Process biochemistry, 46(2), 505-511. Obtenido de http://www.sciencedirect.com/science/article/pii/S1359511310003843
Tripathi, G. (2009). Enzyme biotechnology. ABD Publishers.
Published
2017-09-29
How to Cite
Castillo, P., Quinchuela, L., Echeverría, P., & Jácome, G. (2017). Extraction purification and characterization of trypsin inhibitors from Andean seeds. Enfoque UTE, 8(4), pp. 53 - 67. https://doi.org/https://doi.org/10.29019/enfoqueute.v8n4.172
Section
General Engineering