Cargando…

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-clottin...

Descripción completa

Detalles Bibliográficos
Autores principales: 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.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10606240/
https://www.ncbi.nlm.nih.gov/pubmed/37893665
http://dx.doi.org/10.3390/foods12203772
_version_ 1785127269129256960
author 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.
author_facet 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.
author_sort Balabova, Dina V.
collection PubMed
description 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.
format Online
Article
Text
id pubmed-10606240
institution National Center for Biotechnology Information
language English
publishDate 2023
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-106062402023-10-28 Biochemical Properties of a Promising Milk-Clotting Enzyme, Moose (Alces alces) Recombinant Chymosin 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. Foods Article 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. MDPI 2023-10-13 /pmc/articles/PMC10606240/ /pubmed/37893665 http://dx.doi.org/10.3390/foods12203772 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
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.
Biochemical Properties of a Promising Milk-Clotting Enzyme, Moose (Alces alces) Recombinant Chymosin
title Biochemical Properties of a Promising Milk-Clotting Enzyme, Moose (Alces alces) Recombinant Chymosin
title_full Biochemical Properties of a Promising Milk-Clotting Enzyme, Moose (Alces alces) Recombinant Chymosin
title_fullStr Biochemical Properties of a Promising Milk-Clotting Enzyme, Moose (Alces alces) Recombinant Chymosin
title_full_unstemmed Biochemical Properties of a Promising Milk-Clotting Enzyme, Moose (Alces alces) Recombinant Chymosin
title_short Biochemical Properties of a Promising Milk-Clotting Enzyme, Moose (Alces alces) Recombinant Chymosin
title_sort biochemical properties of a promising milk-clotting enzyme, moose (alces alces) recombinant chymosin
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10606240/
https://www.ncbi.nlm.nih.gov/pubmed/37893665
http://dx.doi.org/10.3390/foods12203772
work_keys_str_mv AT balabovadinav biochemicalpropertiesofapromisingmilkclottingenzymemoosealcesalcesrecombinantchymosin
AT belashekaterinaa biochemicalpropertiesofapromisingmilkclottingenzymemoosealcesalcesrecombinantchymosin
AT belenkayasvetlanav biochemicalpropertiesofapromisingmilkclottingenzymemoosealcesalcesrecombinantchymosin
AT shcherbakovdmitryn biochemicalpropertiesofapromisingmilkclottingenzymemoosealcesalcesrecombinantchymosin
AT belovalexandern biochemicalpropertiesofapromisingmilkclottingenzymemoosealcesalcesrecombinantchymosin
AT kovalanatolyd biochemicalpropertiesofapromisingmilkclottingenzymemoosealcesalcesrecombinantchymosin
AT mironovaannav biochemicalpropertiesofapromisingmilkclottingenzymemoosealcesalcesrecombinantchymosin
AT bondaralexandera biochemicalpropertiesofapromisingmilkclottingenzymemoosealcesalcesrecombinantchymosin
AT volosnikovaekaterinaa biochemicalpropertiesofapromisingmilkclottingenzymemoosealcesalcesrecombinantchymosin
AT arkhipovsergeyg biochemicalpropertiesofapromisingmilkclottingenzymemoosealcesalcesrecombinantchymosin
AT sokolovaolgao biochemicalpropertiesofapromisingmilkclottingenzymemoosealcesalcesrecombinantchymosin
AT chirkovavarvaray biochemicalpropertiesofapromisingmilkclottingenzymemoosealcesalcesrecombinantchymosin
AT elchaninovvadimv biochemicalpropertiesofapromisingmilkclottingenzymemoosealcesalcesrecombinantchymosin