ABSTRACT
This study was conducted for investigating expression and enzymatic characteristics of recombinant Oryctolagus cuniculus chymosin (ROCC) expressed in Pichia pastoris. SDS-PAGE of partially purified supernatant displayed two distinct molecular bands approximately at the sizes of 40 kDa and 45 kDa corresponding to chymosin and partially glycosylated chymosin, respectively. Proteolysis assay demonstrated that rabbit chymosin was more specific compared to bovine and camel chymosins when it comes to hydrolyzing α, ß, and κ-casein. Rabbit chymosin kept its stability in a wide pH range (3.0-6.0) at 37 °C for 8 h. Active chymosin exhibited maximum enzymatic activity at 40 °C and pH 4.0 with the addition of 75 mM CaCl2. The ROCC clotting activity on donkey, cow, goat, lamb, camel milk was determined as 40, 10, 5.7, 3.07, and 2.66 IMCU/mL, respectively. These results revealed that ROCC might possess a potential for incorporation into cheese manufacture technology as a milk-clotting enzyme.
Subject(s)
Chymosin , Gene Expression , Saccharomycetales , Animals , Chymosin/biosynthesis , Chymosin/chemistry , Chymosin/genetics , Rabbits , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Saccharomycetales/genetics , Saccharomycetales/metabolismABSTRACT
Calf rennet has long been used in cheese-making. Because of calf rennet shortage and high cost, novel proteases were needed to meet industry's increasing enzyme demand. Recombinant chymosins and camel chymosin were started to be used in the industry. There is no study in the literature subjecting use of rabbit rennet in cheese production. Chemical, rheological, and sensorial characteristics of white cheese made with rabbit rennet were investigated in this study. Quality characteristics of rabbit rennet cheese (RC) were compared to cheeses produced with commercial calf (CC) and camel chymosins (CLC). RC and CLC exhibited higher hardness and dynamic moduli values throughout the storage as compared to CC. Although moisture levels of cheese samples were similar at day 60, CC had much lower hardness and dynamic moduli values than CLC and RC. While the appearance and structure were better for CLC, the highest odor and taste scores were obtained by RC during 60 days of storage. The results of this investigation proposed that rabbit rennet could be a suitable milk coagulant for white cheese production. Our results showed that rabbit rennet has comparable cheese-making performance with camel chymosin and could be a good alternative for calf chymosin.