Recombinant chymosins of musk ox and takin were obtained in the expression system of yeast Kluyveromyces lactis. It was found that according to the parameters of thermal stability, the dependence of milk-clotting activity on pH and the concentration of CаСl2 in milk, recombinant chymosins of takin and musk ox were similar to recombinant cow chymosin, but markedly different from recombinant chymosin of a single-humped camel. The studied biochemical properties of recombinant chymosins of takin and musk ox allow us to consider them as a potential alternative to modern commercial genetically engineered milk coagulants for cheesemaking.

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.

Two recombinant integration vectors containing the maral prochymosin gene under the control of various yeast promoters have been developed. The milk-clotting activity of the culture medium of the producer (Kluyveromyces lactis) containing the expression cassette with the GAP promoter was 488,3±3,5 AU/ml, with the TEF promoter – 249,0±2,4 AU/ml.

The review discusses the basic biochemical properties of the known recombinant chymosins that are currently used in cheesemaking or may potentially be used as industrial milk coagulants. The parameters of the Michaelis–Menten kinetics, milk-clotting activity, proteolytic activity, specificity, and the dependence of coagulation ability on pH and calcium-ion concentration are considered.

The paper reports on the production of the maral recombinant chymosin in the Escherichia coli expression system (SHaffle express strain) and the study of its biochemical properties relevant for the cheese-making industry. The highest maral recombinant prochymosin content in inclusion bodies was observed when producer cells were cultured at 25°C for 6 h after the introduction of an inducer, 10 mM isopropyl-β-D-1-thiogalactopyranoside. The biochemical properties of the obtained enzyme were compared with those of bovine and dromedarian recombinant chymosins. It is shown that total proteolytic activity of the maral recombinant chymosin was comparable with that of the bovine enzyme and that it exceeded the activity of the dromedarian enzyme by about 3.8 times. The thermal stability of the recombinant chymosin from maral was found to be 5–10°C higher than that of the chymosins from the cow and dromedary. The patterns of dependence of milk-clotting activity on the pH level and calcium chloride concentration in cow’s milk on the enzyme from maral met the requirements set by the cheese industry. Its high proteolytic activity and thermal stability limits the scope of application of maral recombinant chymosin to the production of cheeses with short ripening and short storage times.

This paper discusses biochemical properties of the recombinant chymosin in alpaca (Vicugnapacos), which influence the production of rennet cheeses. These properties determine its value in the production of rennet cheeses. Recombinant bovine chymosins are used as a control. In comparison to them, the recombinant alpaca chymosin is characterized by a high specificity towards bovine κ-casein: the threshold for its thermal inactivation is 10–15°C higher and reaches 60°C. The nature of the relation of its specific activity to the pH and concentration of CaCl meets the requirements for the use of milk-clotting enzymes in the production of rennet cheeses.

Рекомбинантные ферменты на сегодняшний день применяются практически во всех сферах деятельности человека. При этом постоянно ведутся работы по усовершенствованию (модификации) существующих ферментов и поиску новых. Рекомбинантный химозин активно используется в пищевой промышленности для производства сыров. В ходе данного исследования гены химозина и прохимозина В теленка, были клонированы в составе экспрессионного вектор pET21a. Было показано, что рекомбинантный химозин накапливается в бактериальной массе гораздо интенсивней, чем прохимозин. При этом препарат прохимозин обладает высокой ферментативной активностью, в то время как химозин не проявляет такой активности.