Identification and purification of a new milk-clotting protease from Onopordum leptolepis

Milk coagulation is the basic step in cheese making. Milk clotting enzymes show the main role in cheese manufacturing ,which their activity leads to the enzyme-mediated cleavage of kappa-casein, rendering the casein micelles unstable and eventually aggregation that yields a clot and a gel afterwards. Chymosin (EC ۳.۴.۲۳.۴) from the calf rennet has been considered as the most widely used milk-clotting enzyme. Increasing world cheese production and consumption along with a reduced supply of calf rennet, has led to a systematic investigation for new rennet substitutes. The results of research conducted in recent years have shown the possibility of using plant proteases as a suitable, low-cost and safe alternative to others rennet. Accordingly, some plant milk-clotting proteases have been isolated from Withania coagulans, Cynara scolymus, Silybum marianum ,Centaurea calcitrapa, Solanum dubium and Asparagus officinalis. Here, a new milk-clotting protease from Onopordum leptolepis was identified and purified. The enzyme was extracted by grinding the flower of the plant in liquid nitrogen, then homogenized using sodium citrate buffer (pH ۳.۰). The plant extract was filtered and the enzyme was purified using ammonium sulfate precipitation. The homogeneity of the enzyme was assessed by SDS-PAGE. The crude enzyme showed the milk clotting activity about ۶۲.۸۵ ± ۳ U/ml when assayed against bovine milk. The highest caseinolytic activity was detected at pH ۳ and ۴۵ °C temperature. The simple purification procedure together with the availability of the plants, as the enzyme-producing source, is cost effective in biotechnology. This approach could be used for large-scale production of the enzyme, allowing a broad study of its various probable applications. Moreover, high milk-clotting ability of the protease, could therefore pave the way for its uses in the cheese industry as well as other food and biotechnological industries.

کلیدواژه ها:

Plant rennet ، Onopordum leptolepis ، Milk-clotting activity