Proteases from Latex of Euphorbia spp. and Its Application on Milk Clot Formation

Fidia Fibriana, Apichat Upaichit

Abstract


Crude proteases were extracted from Euphorbiaceae family, i.e. E. milii var imperata, E. trigona, and E. maculata. Among those three crude proteases, the activity of protease from E. trigona was the highest (812.50 U/ml), whereas E. milii and E. maculata crude proteases activity were 298.60 U/ml and 95.80 U/ml, respectively. E. maculata protein concentration was the highest among those three crude enzymes (1.206 mg/ml). The optimum pH and temperature of the enzymes were pH 7.0, pH 6.0, pH 6.5 and 60 C, 50 C, and 50 C, respectively. Crude protease from E. milii var imperata, E. trigona, and E. maculata retained proteolytic activity over a wide range of pH (5.09.0) and temperature (up to 65 C) with casein as substrate. All crude proteases showed milk clotting activity ranged from 0.58 U/ml to 1.01 U/ml. Thus, these crude proteases are potential to be applied in dairy industries. However, further study on enzyme purification and characterization are necessary to obtain high purity of proteases before its application.

Protease kasar berhasil diekstrak dari tanaman family Euphorbiaceae, yaitu E. milii var imperata, E. trigona, dan E. maculata. Diantara ketiga protease tersebut, aktivitas protease tertinggi diperoleh dari E. trigona (812,50 U/ml), sedangkan aktivitas protease dari E. milii dan E. maculata adalah 298,60 U/ml dan 95,80 U/ml, berturut-turut. Konsentrasi total protein tertinggi terdapat pada protease kasar E. maculata (1,206 mg/ml). pH dan suhu optimum ketiga enzim tersebut adalah pH 7.0, pH 6.0, pH 6.5 dan suhu 60 C, 50 C, and 50 C, berturut-turut. Protease kasar dari E. milii var imperata, E. trigona, dan E. maculata menunjukkan aktivitas proteolitik pada rentang pH 5.09.0 dan rentang suhu sampai 65 C menggunakan kasein sebagai substrat. Semua protease kasar menunjukkan aktivitas penggumpalan susu dengan rentang dari 0,58 U/ml sampai 1,01 U/ml. Berdasarkan hasil yang diperoleh, protease kasar dari ketiga jenis tanaman ini berpotensi untuk diaplikasikan dalam industri olahan susu. Meskipun demikian, studi lanjut mengenai purifikasi dan karakterisasi sangat diperlukan untuk memperoleh protease murni sebelum aplikasi dalam industri makanan, khususnya pada industri olahan susu.


Keywords


plant protease, Euphorbia,

Full Text:

PDF

References


Antao, C.M. and Malcata, F.X. (2005). Plant serine proteases: biochemical, physiological and molecular features. Plant Physiol. Biochem. 43: 637-650

Abubakar, E.M. (2009). Antibacterial activity of crude extracts of Euphorbia hirta against some bacteria associated with enteric infections. J. Med. Plants Res. 3 (7): 498-505.

Arima, K., Uchikoba, T., Yonezawa, H., Shimada, M. and Kaneda, M. (2000). Cucumisin like protease from the latex of Euphorbia supina. Phytochem. 53: 639644.

Badgujar, S.B. and Mahajan, R.T. (2009). Proteolytic enzymes of some laticiferous plants belonging to Khandesh region of Maharashtra, India. J. Phar. Res. 2 (9): 1434-1437

Bigoniya, P. and Rana, A.C. (2009). Radioprotective and In-Vitro Cytotoxic Sapogenin from Euphorbia neriifolia (Euphorbiaceae) Leaf. Trop. J. Pharm. Res. 8 (6): 521-530.

Bradford, M.M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.

Costa, J.O., Kelly, C.F., Mrio, S.G.F, Cleine, C.C., Mariana, V., Heliane, S.S., Renata, B., Nilson, P.S., and Fbio, O. (2010). Structural and functional comparison of proteolytic enzymes from plant latex and snake venoms. Biochimie. 92: 1760-1765.

Dahot, M.U., Khan, M.Y. and Memon, A.N. (1990). Screening of some Pakistani plants for milk clotting activity. J. Islamic Acad. Sci. 3 (4): 284-286

Demir, Y., Alayli, A., Yildirim, S. and Demir, N.(2005). Identification of protease from Euphorbia amygdaloides latex and its use in cheese production. Prep. Biochem. Biotechnol. 35: 291299.

Domsalla, A, and Melzig, M.F. (2008). Occurrence and properties of proteases in plant lattices. Planta Med. 74: 699711.

Dubey, V.K., Pande, M., Singh, B.K. and Jagannadham, M.V. (2007). Papain-like proteases: applications of their inhibitors. Afr. J. Biotechnol. 6 (9): 1077-1086.

Esser, H.J. (2010). Flora of Thailand (Euphorbiaceae). National Herbarium Nederland. On line at http://www.nationaalherbarium.nl/thaieuph/ThEspecies/ThEuphorbiaT.htm

Fonseca, K.C., Morais, N.C.G., Queiros, M.R., Silva, M.C., Gomes, M.S., Costa, J.O., Mamede, C.C.N, Torres, F.S., Penha-Silva, N., Beletti, M.E., Canabrava, H.A.N. and Oliviera, F. (2010). Purification and biochemical characterization of Eumiliin from Euphorbia milii var. hislopii latex. Journal of Phytochemistry. 71, 708-715.

Genelhu, M.S., Zanini, M.S., Voloso, I.F., Carneiro, A.M.D., Lopes, M.T.P. and Salas, C.E. (1998). Use of a cysteine proteinase from Carica candamarcensis as a protective agent during DNA extraction. Brazil. J. Med. Biol. Res. 31: 1129-1132

Hagel, J.M., Yeung, E.C. and Facchini, P.J. (2008). Got milk? The secret life of laticifers. Trends Plant Science. 13, 631639.

Kumar, C.G. and Takagi, H. (1999). Microbial alkaline proteases: from bioindustrial viewpoint. Biotechnol Adv. 17: 561-594

Ko, J. H., Chow K. S. and Han K. H. (2003). Transcriptome analysis reveals novel features of the molecular events occurring in the laticifers ofHevea brasiliensis(para rubber tree). Plant Mol. Biol. 53 (4): 479492.

Konno, K. (2011). Plant latex and other exudates as plant defense systems: Roles of various defense chemicals and proteins contained therein. Phytochem. 72: 15101530.

Kupchan, S.M., Uchida I., Branfman A.R., Dailey R.G. Jr., and Fei B.Y. (1976). Antileukemic principles isolated from euphorbiaceae plants. Science. 191: 571-572.

Leary, D., Marjo, V., Gwenae, H., Salvatore, A., and Catherine M. (2009). Marine genetic resources: A review of scientific and commercial interest. Mar. Policy. 33: 183 194.

Mahajan, R.T. and Badgujar, S.B. (2010). Biological aspects of proteolytic enzymes: A Review. J. Pharm. Res. 3 (9): 2048-2068.

Natarajan, D., Nagamurugan, N., Ramachandran, A., Mohanasundari C., Srinivasan, K. (2007). Anticandidial and anticryptococcal activity of Euphorbia fusiformis, a rare medicinal plant. World J. Microbiol. Technol. 23: 719-721.

Naveena, B.M., Mendiratta, S.K., and Anjaneyulu, A.S.R. (2004). Tenderization of buffalo meat using plant proteases from Cucumis trigonus Roxb (Kachri) and Zingiber officinale roscoe (Ginger rhizome). Meat Sci. 68(3): 363369.

Okwu, D.E.(2001). Evaluation of the chemical composition of indigenous Spices and flavouring Agents. Global J. Pure Appl. Sci. 7(3): 455-459.

Patel, A.K., Singh, V.K., and Jagannadham, M.V. (2007). Carnein, a Serine Protease from Noxious Plant Weed Ipomoea carnea (Morning Glory). J. Agric. Food Chem. 55 (14): 5809 -5818 .

Pereira, D.A., Ramos, M.V., Souza, D.P., Portela, T.C.L., Guimar?es, J.A., Madeira, S.V.F., Moussaoui, A.E., Nijs, M., Paul, C., Wintjens, R., Vincentelli, J., Azarkan, M., and Looze, Y. (2001). Revisiting the enzymes stored in the laticifers of Carica papaya in the context of their possible participation in the plant defence mechanism. Cell. Mol. Life Sci. 58: 556570.

Ramezani, R., Aminlari, M. and Fallahi, H. (2003). Effect of chemically modified soy proteins and ficin-tenderized meat on the quality attributes of sausage. J. Food Sci. 68(1): 85-88.

Rawlings, N.D., Alan, J.B., and Alex, B. (2010). MEROPS: the peptidase database. Nucleic Acids Res. 38.

Scopes R.K. (1993). Protein Purification: Principles and Practice. Springer: USA.

Silva, J.R.A., Amaral, A.C.F., Siani, A.C., Rezende, C.M., Felcman, J. and Pinto, A.C. (2003). Contribution to the study of Himatanthus sucuuba latex macromolecule, microelements and carbohydrate. Acta Amazonica 33 (1): 105-110.

Siota, L.F. and Villa, T.G. (2011). Native and biotechnologically engineered plant proteases with industrial application. Food Bioprocess Technol. 4: 10661088.

Tomar, R., Kumar, R., and Jagannadham, M.V. (2008). A Stable SerineProtease, Wrightin, from the Latex of the Plant Wrightia tinctoria Roxb. R. Br.: Purification and Biochemical Properties. J. Agric. Food Chem. 56(4): 1479-1487.

Wang, W., Fuju, T. and Shaoning, C. (2008). Optimizing protein extraction from plant tissues for enhanced proteomics analysis. J. Sep. Sci. 31: 2032-2039.

Yadav, S.C., Pande, M., and Jagannadham, M.V. (2006). Highly stable glycosylated serine protease from the medicinal plant Euphorbia milii. Phytochem. 67 (14): 1414-1426.

Yadav, R.P., Patel A.K., and Jagannadham, M.V. (2011). Purification and biochemical characterization of a chymotrypsin-like serine protease from euphorbia neriifolia Linn. Process Biochem. 46: 1654-1662.




DOI: https://doi.org/10.15294/biosaintifika.v7i2.3951

Refbacks

  • There are currently no refbacks.




Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.