Fmoc-Ser(tBu)-OH Proteases as multifunctional enzymes play
Proteases, as multifunctional enzymes, play key roles in controlling a wide variety of cellular and extracellular processes in plants and animals, including protein degradation and processing, germination, complement activation, inflammation, responses to environmental stimuli, senescence and so on. Complex processes governing either the normal physiology of the cell or abnormal pathophysiological conditions could be more clarified by considering the role of these Fmoc-Ser(tBu)-OH as well . Proteases account for approximately 60% of global markets for enzymes, with a quite wide range of applications in food, detergent and pharmaceutical industries . Most plant proteases are active over wide ranges of temperatures, pH, surfactants, solvents and denaturing agents. Therefore, scientists have directed their research for investigation of these type proteolytic enzymes . A large variety of proteases are used in detergent industry due to their good performance in cleaning the spots and dirt. They have also been utilized in many food applications including meat tenderization, bakery, and preparation of protein hydrolysates . More importantly, they have been used in dairy industry for cheese manufacturing, where they destabilize casein micelles and stimulate milk-clotting, as the main step for cheese production. The specific cleavage of κ-casein at the peptide bond between Phe105-Met106 provokes micelle destabilization and eventually causes aggregation and transformation of the milk into the clot and whey phases . Chymosin (EC 188.8.131.52), is one of the main enzymatic compounds present in calf rennet with considerable and specific proteolytic activity. It is known as the most suitable coagulant with a high milk-clotting/proteolytic activity ratio . It is worth reminding that calf rennet is quite expensive with a limited production rate and reduced supply. Therefore, a need for alternative milk coagulating enzymes is rational considering the demanding market for cheese. To cover this aim, a number of enzymes have been isolated and studied from an animal, microbial and plant sources . Although microbial proteases produced by genetically engineered bacteria have been proven as calf rennet substitutes, the public debate on genetically engineered foods makes their use challenging. Besides in some nations, due to factors such as religion and food habits, production of rennet from animal sources is highly restricted. On the other hand, animal and microbial sources of rennets are limited and do not meet the worldwide growing demands. Therefore, a significantly high research attention has been directed towards plant coagulants . Accordingly, some plant milk-clotting proteases have been isolated from Withania coagulans, Cynara scolymus, Silybum marianum, Centaurea calcitrapa, Oryza sativa, Solanum dubium and Asparagus officinalis . However, drawbacks including bitter taste, poor cheese yield, and defects in flavor and texture of some plant coagulants have limited their use in cheese manufacturing. Thus, a continuing search for alternative plant milk-clotting proteases is still needed . In Portugal and some regions of Spain, coagulants from plant sources, such as an aqueous extract from Cynara cardunculus flowers have been routinely used as the rennet source for the preparation of Mato, Serra and traditional cheeses . The largest groups of plant-derived proteases are from cysteine family of proteases with a wide range of industrial applications including cheese production and meat processing. It has been reported that in-vitro synthesis of peptides takes advantages from the catalytic action of cysteine proteases . Cysteine proteases such as papain from Carica papaya, actinidain from Actinidia chinensis, zingibain from Zingiber officinale and bromelain from Ananas comosus  and Nivulian-II from Euphorbia nivulia  are widely used in several food industries. A considerable resistance over a wide range of temperature and pH makes this group of proteases suitable for commercial uses. Some plants within the family Moraceae such as Ficus carica sylvestris, Ficus religiosa and Ficus racemosa  have been reported as sources of milk-clotting enzymes. Ficus johannis Boiss. as an indigenous plant in the dry, hilly, rocky grounds of Iran, Pakistan, and Afghanistan, is traditionally used for turning milk into cheese, especially in rural areas. In the present research, a milk-clotting cysteine protease from the latex of F. johannis was purified and its biochemical properties were evaluated, aiming to introduce a suitable route for its application in dairy industry.