Isolation and characterization of Cysteine protease from leguminous cotyledons


  • Ranajit Kumar Shaha Department of Bio-industrial Technology, Faculty of Agro Based Industry, University Malaysia Kelantan, Jeli Campus, 17600 Jeli, Kelantan, Malaysia.
  • Nurul Azurin Binti Badruzaman Department of Biochemistry and Molecular Biology, University of Rajshahi, Rajshahi, Bangladesh.
  • Asrul Afandi Department of Biochemistry and Molecular Biology, University of Rajshahi, Rajshahi, Bangladesh.
  • Shyam Sundar Shaha Bangladesh Institute of Health Science Hospital, Department of Immunology, 125/1 Darussalam Mirpur, Dhaka-1216, Bangladesh.


Protease activity, Characterization, Industrial purpose, Leguminous seeds


Proteolytic enzymes play central role in the biochemical mechanism of germination and intricately involved in many aspects of plant physiology and development. The present study was conducted on the compares studies the Cysteine proteases from four varieties of 62 hours germinated leguminous seeds: lentil, green gram, black gram and pea bean. We elaborated the easy procedure for isolation of protease from leguminous germinated seeds by using (NH4)2SO4 precipitation followed by Gel-filtration and DEAE-cellulose chromatography from the 72h germinated cotyledons of lentil (Lens esculenta), green gram (Vigna radiata), black gram (Vigna mungo) and pea bean (Phaseolus vulgaris). This study revealed that the water-soluble protein concentration of crude extract ranged between 2.03 to 2.36mg/ml in which green gram was highest protein concentration (2.36mg/ml) and lentil accounted was the lowest concentration (2.03mg/ml). Cysteine proteases from all different leguminous seeds show very close monomer with a molecular mass of 29.5–30kDa were determined by SDS-PAGE. The enzyme activities were completely inhibited by pCMB, iodoacetate and DEPC indicating Cysteine and histidine residues at the active site. The enzyme is fairly stable towards pH and temperature. Cysteine protease has broad substrate specificity and stability in pH, temperature, therefore, this protease may turn out an efficient choice for the food, pharmaceutical, medicinal, and biotechnology industries.


Download data is not yet available.


Al-Shehri, M.A. & Mostafa S.Y. (2004). Production and some properties of protease produced byBacillus licheniformis isolated from Tihamet Aseer, Saudi Arabia. Pakistan J. Biol. Sci., 7(9): 1631–1635.

Sandhya, C., Sumantha, A. & Pandey, A. (2004). Proteases. In: Pandey, A., Webb, C., Soccol, C.R. & Larroche, C. (eds), Enzyme Technology. Asiatech Publishers, Inc., New Delhi, pp. 319-332.

Smit, G., Smit, B.A. & Engels, W.J.M. (2005). Flavour formation by lactic acid bacteria and biochemical flavour profiling of cheese products. FEMS Microbiol. Rev., 29(3): 591–610.

Wang, L. & Wang, Y.-J. (2004). Rice starch isolation by neutral protease and high-intensity ultrasound. J. Cereal Sci., 39(2): 291–296.

Soper, R. (1998). Biological Science (low price ed). Cambridge University Press, pp. 507-510.

Najafi, M.F., Deobagkar, D. & Deobagkar, D. (2005). Potential application of protease isolated from Pseudomonas aeruginosa PD100. Electron. J. Biotechnol., 8(2): 197–203.

Uhlig, H. (1998). Industrial Enzymes and Their Applications. John Wiley & Sons, Inc., New York, pp. 146-151.

Schaller, A. (2004). A cut above the rest: the regulatory function of plant proteases. Planta, 220(2): 183–197.

Müntz, K., Belozersky, M.A., Dunaevsky, Y.E., Schlereth, A. & Tiedemann, J. (2001). Stored proteinases and the initiation of storage protein mobilization in seeds during germination and seedling growth. J. Exp. Bot., 52(362): 1741–1752.

Rotari, V., Senyuk, V., Horstmann, C., Jivotovskaya, A. & Vaintraub, I. (1997). Proteinase A-like enzyme from germinated kidney bean seeds. Its action on phaseolin and vicilin. Physiol. Plant., 100(1): 171–177.

Tiedemann, J., Neubohn, B. & Müntz, K. (2000). Different functions of vicilin and legumin are reflected in the histopattern of globulin mobilization during germination of vetch (Vicia sativa L.). Planta, 211(1): 1–12.

He, F., Huang, F., Wilson, K.A. & Tan-Wilson, A. (2007). Protein storage vacuole acidification as a control of storage protein mobilization in soybeans. J. Exp. Bot., 58(5): 1059–1070.

Jinka, R. & Rao, P.R. (2002). Storage protein degradation in germinating horse gram seeds. Ind. J. Plant Physiol., 7: 314-320.

Ramakrishna, V. & Ramakrishna Rao, P. (2006). Storage protein degradation in germinating Indian bean (Dolichos lablab L. var. lignosus) seeds. Seed Sci. Technol., 34(1): 161–168.

Zakharov, A., Carchilan, M., Stepurina, T., Rotari, V., Wilson, K. & Vaintraub, I. (2004). A comparative study of the role of the major proteinases of germinated common bean (Phaseolus vulgaris L.) and soybean (Glycine max (L.) Merrill) seeds in the degradation of their storage proteins. J. Exp. Bot., 55(406): 2241–2249.

Shutov, A.D. & Vaintraub, I.A. (1987). Degradation of storage proteins in germinating seeds. Phytochemistry, 26(6): 1557–1566.

Jones, B.L. (2005). Endoproteases of barley and malt. J. Cereal Sci., 42(2): 139–156.

Rotari, V., Senyuk, V., Horstmann, C., Jivotovskaya, A. & Vaintraub, I. (1997). Proteinase A-like enzyme from germinated kidney bean seeds. Its action on phaseolin and vicilin. Physiol. Plant., 100(1): 171–177.

Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193(1): 265–275.

Sarath, G., Dela motte, R.S. & Wagner, F.W, (1989). Proteolytic Enzyme. A Practical Approach. IRL Press: Oxford, England.

Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. 2nd ed., NY: Cold Spring Harbor Laboratory Press.

Rosen, H. (1957). A modified ninhydrin colorimetric analysis for amino acids. Arch. Biochem. Biophys., 67(1): 10–15.

Dahot, M.U. (1992). Investigation of Proteases in Plant Seeds. Med. J. Islamic World Acad. Sci., 5(4): 241-244.

Beilinson, V., Moskalenko, O.V., Livingstone, D.S., Reverdatto, S.V., Jung, R. & Nielsen, N.C. (2002). Two subtilisin-like proteases from soybean. Physiol. Plant., 115(4): 585–597.

Devaraj, K.B., Gowda, L.R. & Prakash, V. (2008). An unusual thermostable aspartic protease from the latex of Ficus racemosa (L.). Phytochemistry, 69(3): 647–655.

Ramakrishna, V. & Rao, P.R. (2005). Purification of acidic protease from the cotyledons of germinating Indian bean (Dolichos lablab L. var lignosus) seeds. Afr. J. Biotechnol., 4(7): 703–707.

Karunagaran, D. & Rao, P.R. (1990). Axial control of protease development in the cotyledons of horse gram (Macrotyloma uniflorum Lam) seeds during germination. Indian J. Plant Physlol., 33(3): 232-238.

Jiang, L. & Rogers, J.C. (1998). Integral membrane protein sorting to vacuoles in plant cells: evidence for two pathways. J. Cell Biol., 143(5): 1183–1199.

Baumgartner, B. & Chrispeels, M.J. (1977). Purification and characterization of vicilin peptidohydrolase, the major endopeptidase in the cotyledons of mung-bean seedlings. Eur. J. Biochem., 77(2): 223–233.

Koehler, S.M. & Ho, T.-H.D. (1990). A major Gibberellic Acid-Induced Barley Aleurone Cysteine Proteinase which Digests Hordein: Purification and Characterization. Plant Physiol., 94(1): 251–258.

Liu, X., Zhang, Z., Barnaby, N., Wilsona, K.A. & Tan-Wilsona, A. (2001). Soybean subtilisin-like protease involved in initiating storage protein degradation. Seed Sci. Res., 11(1): 55–68.

Dunn, M.J. (1989). Protein purification methods: A practical approach. Edited by Harris, E.L.V. & Angel, S., IRL Press at Oxford University Press, Oxford.

Seo, S., Tan-Wilson, A. & Wilson, K.A. (2001). Protease C2, a cysteine endopeptidase involved in the continuing mobilization of soybean beta-conglycinin seed proteins. Biochim. Biophys. Acta., 1545(1-2): 192–206.


Abstract views: 27 / PDF downloads: 9



How to Cite

Shaha, R. K., Badruzaman, N. A. B., Afandi, A., & Shaha, S. S. (2013). Isolation and characterization of Cysteine protease from leguminous cotyledons. Advances in BioScience, 4(2), 63–73. Retrieved from




Most read articles by the same author(s)