Exploring the Milk-Clotting and Proteolytic Activities in Different Tissues of Vallesia glabra: a New Source of Plant Proteolytic Enzymes.

Publisher: Humana Press Country of Publication: United States NLM ID: 8208561 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1559-0291 (Electronic) Linking ISSN: 02732289 NLM ISO Abbreviation: Appl Biochem Biotechnol Subsets: MEDLINE

Original Publication: Clifton, N.J. : Humana Press, c1981-

Proteolysis*; Apocynaceae/*enzymology ; Milk/*chemistry ; Peptide Hydrolases/*chemistry ; Plant Leaves/*enzymology ; Plant Proteins/*chemistry ; Seeds/*enzymology; Animals ; Hydrogen-Ion Concentration

Proteolytic enzymes are widely distributed in nature, playing essential roles in important biological functions. Recently, the use of plant proteases at the industrial level has mainly increased in the food industry (e.g., cheesemaking, meat tenderizing, and protein hydrolysate production). Current technological and scientific advances in the detection and characterization of proteolytic enzymes have encouraged the search for new natural sources. Thus, this work aimed to explore the milk-clotting and proteolytic properties of different tissues of Vallesia glabra. Aqueous extracts from the leaves, fruits, and seeds of V. glabra presented different protein profiles, proteolytic activity, and milk-clotting activity. The milk-clotting activity increased with temperature (30-65 °C), but this activity was higher in leaf (0.20 MCU/mL) compared with that in fruit and seed extracts (0.12 and 0.11 MCU/mL, respectively) at 50 °C. Proteolytic activity in the extracts assayed at different pH (2.5-12.0) suggested the presence of different types of active proteases, with maximum activity at acidic conditions (4.0-4.5). Inhibitory studies indicated that major activity in V. glabra extracts is related to cysteine proteases; however, the presence of serine, aspartic, and metalloproteases was also evident. The hydrolytic profile of caseins indicated that V. glabra leaves could be used as a rennet substitute in cheesemaking, representing a new and promising source of proteolytic enzymes.

Rawlings, A. N. D., & Barrett, A. J. (1993). Evolutionary families of peptidases. Biochemical Journal., 290(1), 205–218. (PMID: 1132403)
Van der Hoorn, R. A. L. (2008). Plant proteases: from phenotypes to molecular mechanisms. Annual Review of Plant Biology, 59(1), 191–223. (PMID: 18257708)
Adamczyk, B., Smolander, A., Kitunen, V., & Godlewski, M. (2012). Proteoid roots and exudation of proteases by plant roots. In J. Vivanco & F. Baluska (Eds.), Secretions and exudates in biological systems (pp. 75–89). Berlin Heidelberg: Springer.
Jisha, V. N., Smitha, R. B., Pradeep, S., Sreedevi, S., Unni, K. N., Sajith, S., Priji, P., Josh, M. S., & Benjamin, S. (2013). Versatility of microbial proteases. Advances in Enzyme Research, 1(3), 39–51.
Feijoo-Siota, L., & Villa, T. G. (2011). Native and biotechnologically engineered plant proteases with industrial applications. Food and Bioprocess Technology, 4(6), 1066–1088.
Mazorra-Manzano, M. A., Ramírez-Suarez, J. C., & Yada, R. Y. (2018a). Plant proteases for bioactive peptides release: a review. Critical Reviews in Food Science and Nutrition, 58(13), 2147–2163. (PMID: 28394630)
Salas, C. E., Gomes, M. T. R., Hernández, M., & Lopes, M. T. P. (2008). Plant cysteine proteinases: evaluation of the pharmacological activity. Phytochemistry, 69(12), 2263–2269. (PMID: 18614189)
López-Expósito, I., Miralles, B., Amigo, L., & Hernández-Ledesma, B. (2017). Health effects of cheese components with a focus on bioactive peptides. In J. Frias, C. Martinez-Villaluenga, & E. Peñas (Eds.), Fermented foods in health and disease prevention (pp. 239–273). New York: Academic Press.
Fox, P. F., Guinee, T. P., Cogan, T. M., & McSweeney, P. L. H. (2017). Fundamentals of cheese science (2nd ed.pp. 185–229). New York: Springer.
Mazorra-Manzano, M. A., Moreno-Hernández, J. M., & Ramírez-Suarez, J. C. (2018b). Milk-clotting plant proteases for cheesemaking. In M. G. Guevara & G. R. Daleo (Eds.), Biotechnological applications of plant Proteolytic enzymes (pp. 21–41). Cham: Springer.
Jacob, M., Jaros, D., & Rohm, H. (2011). Recent advances in milk clotting enzymes. International Journal of Dairy Technology, 64(1), 14–33.
Veríssimo, P., Faro, C., Moir, A. J. G., Lin, Y., Tang, J., & Pires, E. (1996). Purification, characterization and partial amino acid sequencing of two new aspartic proteinases from fresh flowers of Cynara cardunculus L. European Journal of Biochemistry, 235(3), 762–768. (PMID: 8654427)
Almeida, C. M., & Simões, I. (2018). Cardoon-based rennets for cheese production. Applied Microbiology and Biotechnology, 102(11), 4675–4686. (PMID: 29696340)
Gimenez, G., & Albornoz, P. (2013). Anatomía foliar de Vallesia glabra (Apycynaceae), especie de importancia medicinal y en frugivoría. Lilloa, 50(1), 25–32.
Bourdy, G., Oporto, P., Gimenez, A., & Deharo, E. (2004). A search for natural bioactive compounds in Bolivia through a multidisciplinary approach: Part VI. Evaluation of the antimalarial activity of plants used by Isoceno-Guaranı Indians. Journal of Ethnopharmacology, 93(2–3), 269–277. (PMID: 15234764)
Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurenment with the folin phenol reagent. Journal of Biological Chemistry, 193, 265–275.
Arima, K., Ya, J., & Iwasaki, S. (1970). Milk-clotting enzyme from Mucor pusillus var. Lindt. In E. G. Pearlman & L. Lorand (Eds.), Methods in enzymology (pp. 446–459). New York: Academic Press.
Kumar, A., Singh, V. K., & Jagannadham, M. V. (2007). Carnein, a serine protease from noxious plant weed Ipomoea carnea (morning glory). Journal of Agricultural and Food Chemistry, 55(5809), 5818.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680–685.
Díaz-López, M., Moyano-López, F. J., Alarcón-López, F. J., García-Carreño, F. L., & del Toro, M. A. N. (1998). Characterization of fish acid proteases by substrate–gel electrophoresis. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 121(4), 369–377.
Mazorra-Manzano, M. A., Moreno-Hernandez, J. M., Ramirez-Suarez, J. C., & Torres-Llanez, M.d.J., Gonzalez-Cordova, A.F., & Vallejo-Cordoba, B. (2013a). Sour orange Citrus aurantium L. flowers: a new vegetable source of milk-clotting proteases. LWT-Food Science and Technology, 54(2), 325–330.
Singh, N., Jain, N., Kumar, R., Jain, A., Singh, N. K., & Rai, V. (2015). A comparative method for protein extraction and 2-D gel electrophoresis from different tissues of Cajanus cajan. Frontiers in Plant Science, 6, 606. (PMID: 263009034528993)
Wang, W., Tai, F., & Chen, S. (2008). Optimizing protein extraction from plant tissues for enhanced proteomics analysis. Journal of Separation Science, 31(11), 2032–2039. (PMID: 18615819)
Schaller, A. (2004). A cut above the rest: the regulatory function of plant proteases. Planta., 220(2), 183–197. (PMID: 15517349)
Tan-Wilson, A. L., & Wilson, K. A. (2012). Mobilization of seed protein reserves. Physiologia Plantarum, 145(1), 140–153. (PMID: 22017287)
Yadav, R. P., Patel, A. K., & Jagannadham, M. V. (2012). Neriifolin S, a dimeric serine protease from Euphorbia neriifolia Linn.: purification and biochemical characterisation. Food Chemistry, 132(3), 1296–1304. (PMID: 29243614)
Jamir, K., & Seshagirirao, K. (2018). Purification, biochemical characterization and antioxidant property of ZCPG, a cysteine protease from Zingiber montanum rhizome. International Journal of Biological Macromolecules, 106, 719–729. (PMID: 28830774)
Shewry, P. R., & Halford, N. G. (2002). Cereal seed storage proteins: structures, properties and role in grain utilization. Journal of Experimental Botany, 53(370), 947–958. (PMID: 11912237)
Sandoval-Oliveros, M. R., & Paredes-López, O. (2013). Isolation and characterization of proteins from chia seeds (Salvia hispanica L.). Journal of Agricultural and Food Chemistry, 61(1), 193–201. (PMID: 23240604)
Bojórquez-Velázquez, E., Lino-López, G. J., Huerta-Ocampo, J. A., Barrera-Pacheco, A., de la Rosa, A. P. B., Moreno, A., & Osuna-Castro, J. A. (2016). Purification and biochemical characterization of 11S globulin from chan (Hyptis suaveolens L. Poit) seeds. Food Chemistry., 192, 203–211. (PMID: 26304339)
Plaxton, W. C. (2019). Avoiding proteolysis during the extraction and purification of active plant enzymes. Plant and Cell Physiology, 60(4), 715–724. (PMID: 30753712)
Salehi, M., Aghamaali, M. R., Sajedi, R. H., Asghari, S. M., & Jorjani, E. (2017). Purification and characterization of a milk-clotting aspartic protease from Withania coagulans fruit. International Journal of Biological Macromolecules, 98, 847–854. (PMID: 28202335)
Rajagopalan, A., & Sukumaran, B. O. (2018). Three phase partitioning to concentrate milk clotting proteases from Wrightia tinctoria R. Br and its characterization. International Journal Of Biological Macromolecules, 118, 279–288. (PMID: 29894788)
Afsharnezhad, M., Shahangian, S. S., & Sariri, R. (2019). A novel milk-clotting cysteine protease from Ficus johannis: purification and characterization. International Journal of Biological Macromolecules, 121, 173–182. (PMID: 30290262)
Shi, Y., Prabakusuma, A. S., Zhao, Q., Wang, X., & Huang, A. (2019). Proteomic analysis of Moringa oleifera Lam. Leaf extract provides insights into milk-clotting proteases. LWT- Food Science and Technology, 109, 289–295.
Mazorra-Manzano, M. A., Perea-Gutiérrez, T. C., Lugo-Sánchez, M. E., Ramírez-Suarez, J. C., Torres-Llanez, M. J., González-Córdova, A. F., & Vallejo-Cordoba, B. (2013b). Comparison of the milk-clotting properties of three plant extracts. Food Chemistry, 141(3), 1902–1907. (PMID: 23870908)
Sarmento, A. C., Lopes, H., Oliveira, C. S., Vitorino, R., Samyn, B., Sergeant, K., & Barros, M. T. (2009). Multiplicity of aspartic proteinases from cynara cardunculus L. Planta, 230(2), 429–439. (PMID: 19488781)
Mazorra-Manzano, M. A., Moreno-Hernández, J. M., Torres-Llanez, M. J., Ramírez-Suarez, J. C., González-Córdova, A. F., & Vallejo-Córdoba, B. (2013c). Hydrolysis of proteins by an enzymatic extract of sour orange (Citrus aurantium) flowers and milk-clotting properties for cheese production. Biotecnia, 15(3), 29–33.
Vorster, B. J., Cullis, C., & Kunert, K. (2019). Plant vacuolar processing enzymes. Frontiers in Plant Science, 10, 479. (PMID: 310317946473326)
Moreno-Hernández, J. M. (2013). Caracterización de la actividad coagulante y proteolítica de extractos de flor de naranjo (Citrus aurantium L.) y purificación parcial de una de sus proteasas. Master Thesis. Centro de Investigación en Alimentación y Desarrollo, A.C., Hermosillo Sonora, México.
García-Magaña, M. L., Gonzalez-Borrallo, J., Montalvo-Gonzalez, E., Rudiño-Piñera, E., Sayago-Ayerdi, S. G., & Salazar-Leyva, J. A. (2018). Isoelectric focusing, effect of reducing agents and inhibitors: partial characterization of proteases extracted from Bromelia karatas. Applied Biological Chemistry, 61(4), 459–467.
Moreno-Hernández, J. M., Hernández-Mancillas, X. D., Coss-Navarrete, E. L., Mazorra-Manzano, M. A., Osuna-Ruiz, I., Rodríguez-Tirado, V. A., & Salazar-Leyva, J. A. (2017). Partial characterization of the proteolytic properties of an enzymatic extract from “Aguama” Bromelia pinguin L. fruit grown in Mexico. Applied Biochemistry and Biotechnology, 182(1), 181–196. (PMID: 27830465)
Nishimura, K., Higashiya, K., Ueshima, N., Abe, T., & Yasukawa, K. (2020). Characterization of proteases activities in Ficus carica cultivars. Journal of Food Science., 85(3), 535–544. (PMID: 32027028)
Shi, Y., Wang, X., & Huang, A. (2018). Proteomic analysis and food-grade enzymes of Moringa oleifer Lam. flower. International Journal of Biological Macromolecules., 115, 883–890. (PMID: 29705109)
Wang, X., Shi, Y., He, R., Li, B., & Huang, A. (2020). Label-free quantitative proteomic analysis of the biological functions of Moringa oleifera seed proteins provides insights regarding the milk-clotting proteases. International Journal of Biological Macromolecules, 144, 325–333. (PMID: 31830451)
de Farias, V. A., da Rocha Lima, A. D., Costa, A. S., de Freitas, C. D. T., da Silva Araújo, I. M., dos Santos Garruti, D., Dos Santos, G. D., de Figueiredo, E. A. T., & de Oliveira, H. D. (2020). Noni (Morinda citrifolia L.) fruit as a new source of milk-clotting cysteine proteases. Food Research international, 127, 108689. (PMID: 31882081)
Luo, J., Xiao, C., Zhang, H., Ren, F., Lei, X., Yang, Z., & Yu, Z. (2018). Characterization and milk coagulating properties of Cynanchum otophyllum Schneid. proteases. Journal of Dairy Science, 101(4), 2842–2850. (PMID: 29428763)
Watanabe, Y., Matsushima, S., Yamaguchi, A., & Shioi, Y. (2009). Characterization and cloning of cysteine protease that is induced in green leaves of barley. Plant Science, 176(2), 264–271.
Beka, R. G., Krier, F., Botquin, M., Guiama, V. D., Donn, P., Libouga, D. G., Mbofung, C. M., Dimitrov, K., Slomianny, M. C., Guillochon, D., & Vercaigne-Marko, D. (2014). Characterization of a milk-clotting extract from Balanites aegyptiaca fruit pulp. International Dairy Journal, 34(1), 25–31.
Gogoi, D., Ramani, S., Bhartari, S., Chattopadhyay, P., & Mukherjee, A. K. (2019). Characterization of active anticoagulant fraction and a fibrin(ogen)olytic serine protease from leaves of Clerodendrum colebrookianum, a traditional ethno-medicinal plant used to reduce hypertension. Journal of Ethnopharmacology, 243, 99–112.
Marino, G., Huesgen, P. F., Eckhard, U., Overall, C. M., Schröder, W. P., & Funk, C. (2014). Family-wide characterization of matrix metalloproteinases from Arabidopsis thaliana reveals their distinct proteolytic activity and cleavage site specificity. Biochemical journal, 457(2), 335–346.
Zhang, Y., Wang, H., Tao, L., & Huang, A. X. (2015). Milk-clotting mechanism of Dregea sinensis Hemsl. protease. Journal of Dairy Science., 98(12), 8445–8453. (PMID: 26506540)
Raskovic, B., Lazic, J., & Polovic, N. (2016). Characterisation of general proteolytic, milk clotting and antifungal activity of Ficus carica latex during fruit ripening. Journal of the Science of Food and Agriculture, 96(2), 576–582. (PMID: 25664689)
Llorente, B. E., Obregón, W. D., Avilés, F. X., Caffini, N. O., & Vairo-Cavalli, S. (2014). Use of artichoke (Cynara scolymus) flower extract as a substitute for bovine rennet in the manufacture of Gouda-type cheese: characterization of aspartic proteases. Food Chemistry, 159, 55–63. (PMID: 24767026)
Kazemipour, N., Salehi, I. M., Valizadeh, J., & Sepehrimanesh, M. (2017). Clotting characteristics of milk by Withania coagulans: proteomic and biochemical study. International Journal of Food Properties, 20(6), 1290–1301.

Keywords: Milk-clotting; Plant tissues; Proteolytic enzymes; Vallesia glabra; Zymography

0 (Plant Proteins)
EC 3.4.- (Peptide Hydrolases)

Date Created: 20201003 Date Completed: 20210531 Latest Revision: 20210531

20221213

10.1007/s12010-020-03432-5

33009584