Can Recombinant Tree Shrew (Tupaia belangeri chinensis) Chymosin Coagulate Cow (Bos taurus) Milk?

Genetically engineered chymosin from the tree shrew (Tupaia belangeri chinensis) has been obtained and partially characterized for the first time. The target enzyme was produced in Escherichia coli, strain BL21(DE3). It was shown that tree shrew recombinant chymosin coagulates cow milk (Bos taurus). The total and specific milk-clotting activity of the obtained enzyme was 0.7–5.3 IMCU/mL and 8.8–16.6 IMCU/mg. The nonspecific proteolytic activity of tree shrew recombinant chymosin in relation to total bovine casein was 30 and 117% higher than that of recombinant chymosin of cow and of single-humped camel respectively. It was found that in comparison with most of the known genetically engineered chymosins, the tree shrew enzyme showed exceptionally low thermal stability. After heating at 45°C, the coagulation ability of tree shrew recombinant chymosin decreased by more than 40%, and at 50°C the enzyme lost more than 90% of the initial milk-clotting activity. The Michaelis constant (K_m), enzyme turnover number (k_cat), and catalytic efficiency (k_cat/K_m) for genetically engineered chymosin from the tree shrew were 6.3 ± 0.1 µM, 11 927 ± 3169 s^–1 and 1968 ± 620 µM^–1 s^–1, respectively. Comparative analysis showed that the primary structure of the chymosin-sensitive site of cow kappa-casein and the supposed similar sequence of tree shrew kappa-casein differed by 75%. The ability of tree shrew recombinant chymosin to coagulate cow’s milk, along with a low thermal stability and high catalytic efficiency with respect to the substrate, imitating the chymosin-sensitive site of cow kappa-casein, suggests that this enzyme is of potential interest for cheese making.