International Journal of Biological Macromolecules 70 (2014) 391–398 Contents lists available at ScienceDirect International Journal of Biological Macromolecules j ourna l h o mepa ge: www.elsevier.com/locate/ijbiomac Nivulian-II a new milk clotting cysteine protease of Euphorbia nivulia latex Shamkant B. Badgujar a,b,∗ , Raghunath T. Mahajan b a Department of Biochemistry, National Institute for Research in Reproductive Health (ICMR), Jehangir Merwanji Street, Parel, Mumbai 400012, Maharashtra, India b Faculty of Science, Department of Biotechnology, Moolji Jaitha College, North Maharashtra University, Jalgaon 425002, Maharashtra, India a r t i c l e i n f o Article history: Received 18 April 2014 Received in revised form 23 June 2014 Accepted 10 July 2014 Available online 17 July 2014 Keywords: Cysteine protease Euphorbia nivulia Milk coagulation a b s t r a c t Nivulian-II, new milk clotting cysteine protease has been purified from the latex of Euphorbia nivu- lia Buch.-Ham. Nivulian-II is a monomeric protein with an apparent molecular mass 43670.846 Da. It presents its optimum activity at pH 6.3 and temperature of 50 ◦ C. The enzyme was strongly inhibited by common thiol-blocking reagents thereby indicating that it belongs to cysteine protease family. Nivulian-II is a type of glycoprotein and its pI is 3.4. The N-terminal amino acid sequence of Nivulian-II is DFPPNTCC- CICC. This sequence showed relatively low homology with several other proteases of Euphorbian plants, suggesting that the isolated enzyme is a new cysteine protease. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Milk coagulation is the basic step in cheese making. Milk clotting enzymes are the primary active agents in cheese manufacturing, which involves the enzyme-mediated cleavage of kapa-casein at the peptide bond Phe (105)-Met (106) that renders the casein micelles unstable and eventually causes aggregation that yields a clot and a gel afterwards [1]. Chymosin (EC 3.4.23.4), i.e. ren- net is a milk clotting enzyme obtained from the fourth stomach of the unweaned calf. A shortage of this enzyme has now occurred partly because of the decrease in general availability of suckling calves as they were left for beef and not killed for veal [2]. Another reason for this shortage is the continuously increasing worldwide demand for cheese and cheese products [3]. The recent growth in cheese industry and the scarcity on calf rennet due to the certain religious and ethnic regulations have stimulated the research for milk clotting enzymes from alternative sources. There are many reports on microbial production of milk clotting enzymes avail- able in literature [4–8]. Fungal rennet produced by strains of Mucor miehi and Mucor pusillus have received wide acceptability and in some countries 50–60% of the market has been replaced by these substitutes for calf rennet [9]. ∗ Corresponding author at: Department of Biochemistry, National Institute for Research in Reproductive Health (ICMR), Jehangir Merwanji Street, Parel, Mumbai 400012, Maharashtra, India. Tel.: +91 9765227042; fax: +91 22 24139412. E-mail address: sham83badgujar@gmail.com (S.B. Badgujar). Some researchers have mentioned the use of vegetable pro- teases of Withania coagulans [10], Benincasa cerifera [11], Zingiber officinale, Asparagus officinalis, Ficus carica, Actinidia chinensis and Ananas comosus [12], Calotropis procera [13], Dieffenbachia maculata [14], Onopordum turcicum [15], Cucumis melo [16], Oryza sativa [17], Centaurea calcitrapa [18], Cynara scolymus [19], Silybum marianum [20], sunflower and albizia seeds [21], and Solanum dubium [22] for cheese formation. Proteinase of dried flower of Cynara cardunculus has been recommended as the rennet source for the preparation of Mato and Serra cheese [23–26] and it has also been employed suc- cessfully in Portugal and some region of Spain for the manufacture of traditional cheese [27]. Recently, we reported the milk clotting activity of 21 latex bear- ing plants belonging to seven different laticiferous families viz., Apocynaceae, Asclepiadaceae, Caricaceae, Convolvulaceae, Euphor- biaceae, Moraceae and Sapotaceae. A common feature of the latex of Euphorbiaceae is the presence of noticeable proteolytic and milk clotting activity [28]. To mention here, out of these 21 plants, six plants of Euphorbiaceae member have been characterized bio- chemically for milk clotting activity. The order of potentiality for these six plants with respect to milk clotting activity is Euphor- bia nivulia > Euphorbia milii > Euphorbia prunifolia > Synadenium grantii > Pedilanthus tithymaloides > Euphorbia hirta. Amongst them, Euphorbia nivulia possesses profound milk clotting activity [28]. Therefore, the aim of this investigation is to evaluate alternative source for milk clotting enzyme from plant origin in order to match the increasing worldwide demand for diversified dairy products such as soft cream cheese. http://dx.doi.org/10.1016/j.ijbiomac.2014.07.022 0141-8130/© 2014 Elsevier B.V. All rights reserved.