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- Title
Biochemical Properties of a Promising Milk-Clotting Enzyme, Moose (Alces alces) Recombinant Chymosin.
- Authors
Balabova, Dina V.; Belash, Ekaterina A.; Belenkaya, Svetlana V.; Shcherbakov, Dmitry N.; Belov, Alexander N.; Koval, Anatoly D.; Mironova, Anna V.; Bondar, Alexander A.; Volosnikova, Ekaterina A.; Arkhipov, Sergey G.; Sokolova, Olga O.; Chirkova, Varvara Y.; Elchaninov, Vadim V.
- Abstract
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 Km, kcat and kcat/Km 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.
- Subjects
COAGULANTS; MOOSE; KLUYVEROMYCES marxianus; CHEESEMAKING; PROTEOLYTIC enzymes; AMMONIUM sulfate; THERMAL stability; BLOOD coagulation
- Publication
Foods, 2023, Vol 12, Issue 20, p3772
- ISSN
2304-8158
- Publication type
Article
- DOI
10.3390/foods12203772