Biochemical Properties of a Promising Milk-Clotting Enzyme, Moose ( Alces alces ) Recombinant Chymosin

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-clott...

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Bibliographic Details
Published in:Foods
Main Authors: Dina V. Balabova, Ekaterina A. Belash, Svetlana V. Belenkaya, Dmitry N. Shcherbakov, Alexander N. Belov, Anatoly D. Koval, Anna V. Mironova, Alexander A. Bondar, Ekaterina A. Volosnikova, Sergey G. Arkhipov, Olga O. Sokolova, Varvara Y. Chirkova, Vadim V. Elchaninov
Format: Article in Journal/Newspaper
Language:English
Published: MDPI AG 2023
Subjects:
recombinant chymosin
moose
milk-clotting activity
thermal stability
proteolytic activity
coagulation specificity
Chemical technology
TP1-1185
Alces alces
Online Access:https://doi.org/10.3390/foods12203772
https://doaj.org/article/b17bc991ef924f16b5ba870c8ca9ee26
Description
Summary: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 A 280 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 Ca 2+ 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 Mg 2+ , Zn 2+ , Co 2+ , Ba 2+ , Fe 2+ , Mn 2+ , Ca 2+ , and Cu 2+ 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.

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