This paper discusses biochemical properties of the recombinant chymosin in alpaca ( Vicugna pacos), which influence the production of rennet cheeses. These properties determine its value in...

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.

Moose (Alces alces) recombinant chymosin with a milk-clotting activity of 86 AU/mL was synthesized in the Kluyveromyces lactis expression system. After precipitation with ammonium sulfate and chromatographic purification, a sample of genetically engineered moose chymosin with a specific milk-clotting activity of 15,768 AU/mg was obtained, which was used for extensive biochemical characterization of the enzyme. The threshold of the thermal stability of moose chymosin was 55 °C; its complete inactivation occurred after heating at 60 °C. The total proteolytic activity of moose chymosin was 0.332 A₂₈₀ units. The ratio of milk-clotting and total proteolytic activities of the enzyme was 0.8. The K_m, k_cat and k_cat/K_m values of moose chymosin were 4.7 μM, 98.7 s⁻¹, and 21.1 μM⁻¹ s⁻¹, respectively. The pattern of change in the coagulation activity as a function of pH and Ca²⁺ concentration was consistent with the requirements for milk coagulants for cheese making. The optimum temperature of the enzyme was 50–55 °C. The introduction of Mg²⁺, Zn²⁺, Co²⁺, Ba²⁺, Fe²⁺, Mn²⁺, Ca²⁺, and Cu²⁺ into milk activated the coagulation ability of moose chymosin, while Ni ions on the contrary inhibited its activity. Using previously published data, we compared the biochemical properties of recombinant moose chymosin produced in bacterial (Escherichia coli) and yeast (K. lactis) producers.

Recombinant chymosins (rСhns) of the cow and the camel are currently considered as standard milk coagulants for cheese-making. The search for a new type of milk-clotting enzymes that may exist in nature and can surpass the existing “cheese-making” standards is an urgent biotechnological task. Within this study, we for the first time constructed an expression vector allowing production of a recombinant analog of moose chymosin in the expression system ofEscherichia coli(strain SHuffle express). We built a model of the spatial structure of moose chymosin and compared the topography of positive and negative surface charges with the correspondent structures of cow and camel chymosins. We found that the distribution of charges on the surface of moose chymosin has common features with that of cow and camel chymosins. However, the moose enzyme carries a unique positively charged patch, which is likely to affect its interaction with the substrate. Biochemical and technological properties of the moose rChn were studied. Commercial rСhns of cow and camel were used as comparison enzymes. In some technological parameters, the moose rChn proved to be superior to the reference enzymes. Сompared with the cow and camel rСhns, the moose chymosin specific activity is less dependent on the changes in CaCl₂concentration in the range of 1–5 mM and pH in the range of 6–7, which is an attractive technological property. The total proteolytic activity of the moose rСhn occupies an intermediate position between the rСhns of cow and camel. The combination of biochemical and technological properties of the moose rСhn argues for further study of this enzyme.