Biochemical Properties of a Promising Milk-Clotting Enzyme, Moose (Alces alces) Recombinant Chymosin | Sciact - CRIS-system of Boreskov Institute of Catalysis

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

Journal

Foods
ISSN: 2304-8158

Output data

Year: 2023, Volume: 12, Number: 20, Article number : 3772, Pages count : 19 DOI: 10.3390/foods12203772

Tags

recombinant chymosin; moose; milk-clotting activity; thermal stability; proteolytic activity; coagulation specificity; Michaelis–Menten kinetics; temperature optimum; calcium chloride concentration; substrate pH

Authors

Balabova Dina V. ¹ , Belash Ekaterina A. ¹ , Belenkaya Svetlana V. ² , Shcherbakov Dmitry N. ^1,2 , Belov Alexander N. ³ , Koval Anatoly D. ³ , Mironova Anna V. ³ , Bondar Alexander A. ⁴ , Volosnikova Ekaterina A. ² , Arkhipov Sergey G. ⁵ , Sokolova Olga O. ⁵ , Chirkova Varvara Yu. ¹ , Elchaninov Vadim V. ³

Affiliations

1	Institute of Biology and Biotechnology, Altai State University, 656049 Barnaul, Russia
2	State Research Center for Virology and Biotechnology “Vector”, Rospotrebnadzor, 630559 Koltsovo, Russia
3	Federal Altai Scientific Center for Agrobiotechnologies, Siberian Research Institute of Cheese Making, 656910 Barnaul, Russia
4	JCF “Genomics”, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia
5	Boreskov Institute of Catalysis, Siberan Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia

Funding (1)

Ministry of Science and Higher Education of the Russian Federation

075-15-2022-263

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.

Balabova D.V. , Belash E.A. , Belenkaya S.V. , Shcherbakov D.N. , Belov A.N. , Koval A.D. , Mironova A.V. , Bondar A.A. , Volosnikova E.A. , Arkhipov S.G. , Sokolova O.O. , Chirkova V.Y. , Elchaninov V.V.
Biochemical Properties of a Promising Milk-Clotting Enzyme, Moose (Alces alces) Recombinant Chymosin
Foods. 2023. V.12. N20. 3772 :1-19. DOI: 10.3390/foods12203772 WOS Scopus РИНЦ AN PMID OpenAlex

Submitted:	Sep 22, 2023
Accepted:	Oct 10, 2023
Published print:	Oct 13, 2023
Published online:	Oct 13, 2023

Web of science:	WOS:001097788900001
Scopus:	2-s2.0-85175175124
Elibrary:	55921024
Chemical Abstracts:	2023:2324057
PMID:	37893665
OpenAlex:	W4387614350

DB	Citing
OpenAlex	4
Scopus	1
Web of science	1
Elibrary	5