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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.

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.

For the first time, the chymosin gene (CYM) of a maral was characterized. Its exon/intron organization was established using comparative analysis of the nucleotide sequence. The CYM mRNA sequence encoding a maral preprochymosin was reconstructed. Nucleotide sequence of the CYM maral mRNA allowed developing an expression vector to ensure production of a recombinant enzyme. Recombinant maral prochymosin was obtained in the expression system of Escherichiacoli [strain BL21 (DE3)]. Total milk-coagulation activity (MCA) of the recombinant maral chymosin was 2330 AU/ml. The recombinant maral prochymosin relative activity was 52955 AU/mg. The recombinant maral chymosin showed 100-81% MCA in the temperature range 30-50°C, thermal stability (TS) threshold was 50°C, and the enzyme was completely inactivated at 70°C. Preparations of the recombinant chymosin of a single-humped camel and recombinant bovine chymosin were used as reference samples. Michaelis–Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) of the recombinant maral chymosin, were 1.18 ± 0.1 µM, 2.68 ± 0.08 s−1 and 2.27± 0.10 µm M−1·s−1, respectively.

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 of Escherichia 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 CaCl2 concentration 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.Эталонными коагулянтами молока для сыроделия в настоящее время считаются рекомбинантные химозины (рХн) коровы и верблюда. Нахождение молокосвертывающих ферментов, способных превзойти эталонные коагулянты молока, является актуальной биотехнологической задачей. Нами сконструирован экспрессирующий вектор, который позволил впервые получить рекомбинантный аналог химозина лося в системе экспрессии Escherichia coli (штамм SHuffle Express). Построена модель пространственной структуры химозина лося, и проведено сравнение топографии положительных и отрицательных поверхностных зарядов с соответствующими структурами химозинов коровы и верблюда. Обнаружено, что распределение зарядов на поверхности химозина лося имеет общие черты с распределением зарядов химозинов коровы и верблюда. Отличительная особенность химозина лося – наличие положительно заряженного поверхностного участка, который, вероятно, влияет на его взаимодействие с субстратом. Исследованы основные биохимические и технологические свойства рХн лося. В качестве ферментов сравнения использовали коммерческие рХн коровы и верблюда. Установлено, что по некоторым технологическим показателям рХн лося превосходил ферменты сравнения. По сравнению с рХн коровы и верблюда специфическая активность рХн лося в меньшей степени зависит от изменения концентрации CaCl2 в диапазоне 1–5 мМ и рН в диапазоне 6–7, что является привлекательным технологическим свойством. По общей протеолитической активности рХн лося занимает промежуточное положение между ферментами коровы и верблюда. Совокупность биохимических и технологических свойств рХн лося свидетельствует о необходимости дальнейшего изучения этого фермента.

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.

Due to the scarcity of natural rennet, in this study we have considered an option of using in cheesemaking a milk-clotting enzyme produced by the Irpex lacteus fungus. We describe main properties of I. lacteus coagulant: milk-clotting activity (МА), overall proteolytic activity (PA), thermal stability, MА dependence on рН level and calcium ions content. Partially purified preparations of I. lacteus milk-clotting enzyme was obtained by salting out and gel-filtration. Technological properties of the I. lacteus coagulant were compared to natural calf rennet and cow pepsin (CP). МА of I. lacteus enzyme amounted to 29.1 ± 0.7 RU/ml, with protein content of 23 mg/ml. The coagulant was completely inactivated at 60○С. Thermal stability of I. lacteus milk-clotting enzyme, MA sensitivity to pH variations and Ca2+ content were comparable to respective parameters of calf rennet and CP. Overall PA of the I. lacteus coagulant exceeded CP and calf rennet activity by 33 and 220 times, respectively. As for enzymatic specificity, the following order was observed: calf rennet (100%) > CP (14.9%) > I. lacteus coagulant (0.5%). These findings suggest that there is a need to increase the MA of I. lacteus coagulant in order to be able to use it in cheesemaking. We have considered chemical, biochemical, and genetic corrective actions applicable to technological properties of microfungal milk coagulants.