vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/VJGB-22-31vavilov-3358Research ArticleМОЛЕКУЛЯРНАЯ И КЛЕТОЧНАЯ БИОЛОГИЯMOLECULAR AND CELL BIOLOGYБиохимические и технологические свойства рекомбинантного химозина лося (Alces alces)Biochemical and technological properties of moose (Alces alces) recombinant chymosinhttps://orcid.org/0000-0002-3925-0862БалабоваД. В.BalabovaD. V.

Барнаул

Barnaul

https://orcid.org/0000-0003-2808-4309РудометовА. П.RudometovA. P.

р. п. Кольцово, Новосибирская область

Koltsovo, Novosibirsk Region

БеленькаяС. В.BelenkayaS. V.

Барнаулр. п. Кольцово, Новосибирская областьНовосибирск

BarnaulKoltsovo, Novosibirsk RegionNovosibirsk

БеловА. Н.BelovA. N.

Барнаул

Barnaul

КовальА. Д.KovalA. D.

Барнаул

Barnaul

https://orcid.org/0000-0001-9181-0487БондарьА. А.BondarA. A.

Новосибирск

Novosibirsk

https://orcid.org/0000-0002-4723-9225БакулинаА. Ю.BakulinaA. Yu.

р. п. Кольцово, Новосибирская областьНовосибирск

Koltsovo, Novosibirsk RegionNovosibirsk

РухловаЕ. А.RukhlovaE. A.

р. п. Кольцово, Новосибирская область

Koltsovo, Novosibirsk Region

https://orcid.org/0000-0002-4355-6055ЕльчаниновВ. В.ElchaninovV. V.

Барнаул

Barnaul

https://orcid.org/0000-0001-8023-4453ЩербаковД. Н.ShcherbakovD. N.

Барнаулр. п. Кольцово, Новосибирская область

BarnaulKoltsovo, Novosibirsk Region

dnshcherbakov@gmail.comАлтайский государственный университетРоссияAltai State UniversityRussian FederationГосударственный научный центр вирусологии и биотехнологии «Вектор» РоспотребнадзораРоссияState Research Center of Virology and Biotechnology “Vector”Russian FederationАлтайский государственный университет; Государственный научный центр вирусологии и биотехнологии «Вектор» Роспотребнадзора; Новосибирский национальный исследовательский государственный университетРоссияAltai State University; State Research Center of Virology and Biotechnology “Vector”; Novosibirsk State UniversityRussian FederationФедеральный Алтайский научный центр агробиотехнологий, Сибирский научно-исследовательский институт сыроделияРоссияFederal Altai Scientific Center for Agrobiotechnology, Siberian Research Institute of Cheese-MakingRussian FederationИнститут химической биологии и фундаментальной медицины Сибирского отделения Российской академии наукРоссияInstitute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of SciencesRussian FederationГосударственный научный центр вирусологии и биотехнологии «Вектор» Роспотребнадзора; Новосибирский национальный исследовательский государственный университетРоссияState Research Center of Virology and Biotechnology “Vector”; Novosibirsk State UniversityRussian FederationАлтайский государственный университет; Государственный научный центр вирусологии и биотехнологии «Вектор» РоспотребнадзораРоссияAltai State University; State Research Center of Virology and Biotechnology “Vector”Russian Federation202203062022263240249Copyright © Балабова Д.В., Рудометов А.П., Беленькая С.В., Белов А.Н., Коваль А.Д., Бондарь А.А., Бакулина А.Ю., Рухлова Е.А., Ельчанинов В.В., Щербаков Д.Н., 20222022Балабова Д.В., Рудометов А.П., Беленькая С.В., Белов А.Н., Коваль А.Д., Бондарь А.А., Бакулина А.Ю., Рухлова Е.А., Ельчанинов В.В., Щербаков Д.Н.Balabova D.V., Rudometov A.P., Belenkaya S.V., Belov A.N., Koval A.D., Bondar A.A., Bakulina A.Y., Rukhlova E.A., Elchaninov V.V., Shcherbakov D.N.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/3358

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.

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.

лосьрекомбинантный химозинмолокосвертывающая активностьбиохимические свойствасыроделиеAlces alcesmooserecombinant chymosinmilk-clotting activitybiochemical propertiescheese-makingAlces alcesThis study was funded by the State Task of the Ministry of Science and Education of the Russian Federation (topic No. FZMW-2020-0002, “Design of recombinant enzyme producers for the cheese-making industry”) and The Russian Foundation for Basic Research (grant number 19-44-220010).ReferencesAkishev Z., Kiribayeva A., Mussakhmetov A., Baltin K., Ramankulov Y., Khassenov B. Constitutive expression of Camelus bactrianus prochymosin B in Pichia pastoris. Heliyon. 2021;7(5):e07137. DOI 10.1016/j.heliyon.2021.e07137.Akishev Z., Kiribayeva A., Mussakhmetov A., Baltin K., Ramankulov Y., Khassenov B. Constitutive expression of Camelus bactrianus prochymosin B in Pichia pastoris. Heliyon. 2021;7(5):e07137. DOI 10.1016/j.heliyon.2021.e07137.Bansal N., Drake M.A., Piraino P., Broe M.L., Harboe M., Fox P.F., McSweeney P.L.H. Suitability of recombinant camel (Camelus dromedarius) chymosin as a coagulant for Cheddar cheese. Int. Dairy J. 2009;19(9):510-517. DOI 10.1016/j.idairyj.2009.03.010.Bansal N., Drake M.A., Piraino P., Broe M.L., Harboe M., Fox P.F., McSweeney P.L.H. Suitability of recombinant camel (Camelus dromedarius) chymosin as a coagulant for Cheddar cheese. Int. Dairy J. 2009;19(9):510-517. DOI 10.1016/j.idairyj.2009.03.010.Belenkaya S.V., Balabova D.V., Belov A.N., Koval A.D., Shcherbakov D.N., Elchaninov V.V. Basic biochemical properties of recombinant chymosins (review). Appl. Biochem. Microbiol. 2020a;56(4):363-372. DOI 10.1134/S0003683820040031.Belenkaya S.V., Balabova D.V., Belov A.N., Koval A.D., Shcherbakov D.N., Elchaninov V.V. Basic biochemical properties of recombinant chymosins (review). Appl. Biochem. Microbiol. 2020a;56(4):363-372. DOI 10.1134/S0003683820040031.Belenkaya S.V., Bondar A.A., Kurgina T.A., Elchaninov V.V., Bakulina A.Yu., Rukhlova E.A., Lavrik O.I., Ilyichev A.A., Shcherbakov D.N. Characterization of the Altai maral chymosin gene, production of a chymosin recombinant analog in the prokaryotic expression system, and analysis of its several biochemical properties. Biochemistry (Mosc.). 2020b;85(7):781-791. DOI 10.1134/S0006297920070068.Belenkaya S.V., Bondar A.A., Kurgina T.A., Elchaninov V.V., Bakulina A.Yu., Rukhlova E.A., Lavrik O.I., Ilyichev A.A., Shcherbakov D.N. Characterization of the Altai maral chymosin gene, production of a chymosin recombinant analog in the prokaryotic expression system, and analysis of its several biochemical properties. Biochemistry (Mosc.). 2020b;85(7):781-791. DOI 10.1134/S0006297920070068.Belenkaya S.V., Rudometov A.P., Shcherbakov D.N., Balabova D.V., Kriger A.V., Belov A.N., Koval A.D., Elchaninov V.V. Biochemical properties of recombinant chymosin in alpaca (Vicugna pacos L.). Appl. Biochem. Microbiol. 2018;54(6):569-576. DOI 10.1134/S0003683818060054.Belenkaya S.V., Rudometov A.P., Shcherbakov D.N., Balabova D.V., Kriger A.V., Belov A.N., Koval A.D., Elchaninov V.V. Biochemical properties of recombinant chymosin in alpaca (Vicugna pacos L.). Appl. Biochem. Microbiol. 2018;54(6):569-576. DOI 10.1134/S0003683818060054.Belenkaya S.V., Shcherbakov D.N., Balabova D.V., Belov A.N., Koval A.D., Elchaninov V.V. Production of maral (Cervus elaphus sibiricus Severtzov) recombinant chymosin in the prokaryotic expression system and the study of the aggregate of its biochemical properties relevant for the cheese-making industry. Appl. Biochem. Microbiol. 2020c;56(6):647-656. DOI 10.1134/S0003683820060034.Belenkaya S.V., Shcherbakov D.N., Balabova D.V., Belov A.N., Koval A.D., Elchaninov V.V. Production of maral (Cervus elaphus sibiricus Severtzov) recombinant chymosin in the prokaryotic expression system and the study of the aggregate of its biochemical properties relevant for the cheese-making industry. Appl. Biochem. Microbiol. 2020c;56(6):647-656. DOI 10.1134/S0003683820060034.Belov A.N., Koval A.D., Avdanina E.A., Elchaninov V.V. Effects of milk clotting preparation on cheese organoleptic indices. Syrodelie i Maslodelie = Cheese Making and Butter Making. 2009;1:22-24. (in Russian)Belov A.N., Koval A.D., Avdanina E.A., Elchaninov V.V. Effects of milk clotting preparation on cheese organoleptic indices. Syrodelie i Maslodelie = Cheese Making and Butter Making. 2009;1:22-24. (in Russian)Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976;72(1-2):248-254. DOI 10.1016/0003-2697(76)90527-3.Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976;72(1-2):248-254. DOI 10.1016/0003-2697(76)90527-3.Chen H., Zhang G., Zhang Y., Dong Y., Yang K. Functional implications of disulfide bond, Cys206−Cys210, in recombinant prochymosin (chymosin). Biochemistry. 2000;39(40):12140-12148. DOI 10.1021/bi000976o.Chen H., Zhang G., Zhang Y., Dong Y., Yang K. Functional implications of disulfide bond, Cys206−Cys210, in recombinant prochymosin (chymosin). Biochemistry. 2000;39(40):12140-12148. DOI 10.1021/bi000976o.Costabel L.M., Bergamini C.V., Pozza L., Cuffia F., Candioti M.C., Hynes E. Influence of chymosin type and curd scalding temperature on proteolysis of hard cooked cheeses. J. Dairy Res. 2015;82(3):375-384. DOI 10.1017/S0022029915000175.Costabel L.M., Bergamini C.V., Pozza L., Cuffia F., Candioti M.C., Hynes E. Influence of chymosin type and curd scalding temperature on proteolysis of hard cooked cheeses. J. Dairy Res. 2015;82(3):375-384. DOI 10.1017/S0022029915000175.D’Incecco P., Limbo S., Hogenboom J., Rosi V., Gobbi S., Pellegrino L. Impact of extending hard-cheese ripening: a multiparameter characterization of Parmigiano Reggiano cheese ripened up to 50 months. Foods. 2020;9(3):268. DOI 10.3390/foods9030268.D’Incecco P., Limbo S., Hogenboom J., Rosi V., Gobbi S., Pellegrino L. Impact of extending hard-cheese ripening: a multiparameter characterization of Parmigiano Reggiano cheese ripened up to 50 months. Foods. 2020;9(3):268. DOI 10.3390/foods9030268.Ersöz F., İnan M. Large-scale production of yak (Bos grunniens) chymosin A in Pichia pastoris. Protein Expr. Purif. 2019;154:126-133. DOI 10.1016/j.pep.2018.10.007.Ersöz F., İnan M. Large-scale production of yak (Bos grunniens) chymosin A in Pichia pastoris. Protein Expr. Purif. 2019;154:126-133. DOI 10.1016/j.pep.2018.10.007.Eskandari M.H., Hosseini A., Zarasvand S.A., Aminlari M. Cloning, expression, purification and refolding of caprine prochymosin. Food Biotechnol. 2012;26(2):143-153. DOI 10.1080/08905436.2012.670829.Eskandari M.H., Hosseini A., Zarasvand S.A., Aminlari M. Cloning, expression, purification and refolding of caprine prochymosin. Food Biotechnol. 2012;26(2):143-153. DOI 10.1080/08905436.2012.670829.Flamm E.L. How FDA approved chymosin: a case history. Nat. Biotechnol. 1991;9(4):349-351. DOI 10.1038/nbt0491-349.Flamm E.L. How FDA approved chymosin: a case history. Nat. Biotechnol. 1991;9(4):349-351. DOI 10.1038/nbt0491-349.Fox P.F., Guinee T.P., Cogan T.M., McSweeney P.L.H. Enzymatic coagulation of milk. In: Fundamentals of Cheese Science. Boston: Springer, 2017;185-229. DOI 10.1007/978-1-4899-7681-9_7.Fox P.F., Guinee T.P., Cogan T.M., McSweeney P.L.H. Enzymatic coagulation of milk. In: Fundamentals of Cheese Science. Boston: Springer, 2017;185-229. DOI 10.1007/978-1-4899-7681-9_7.Gumus P., Hayaloglu A.A. Effects of blends of camel and calf chymosin on proteolysis, residual coagulant activity, microstructure, and sensory characteristics of Beyaz peynir. J. Dairy Sci. 2019;102(7):5945-5956. DOI 10.3168/jds.2018-15671.Gumus P., Hayaloglu A.A. Effects of blends of camel and calf chymosin on proteolysis, residual coagulant activity, microstructure, and sensory characteristics of Beyaz peynir. J. Dairy Sci. 2019;102(7):5945-5956. DOI 10.3168/jds.2018-15671.Harboe M., Broe M.L., Qvist K.B. The production, action and application of rennet and coagulants. In: Law B.A., Tamime A.Y. (Eds.) Technology of Cheesemaking. Chichester: Wiley-Blackwell, 2010;98-129. DOI 10.1002/9781444323740.ch3.Harboe M., Broe M.L., Qvist K.B. The production, action and application of rennet and coagulants. In: Law B.A., Tamime A.Y. (Eds.) Technology of Cheesemaking. Chichester: Wiley-Blackwell, 2010;98-129. DOI 10.1002/9781444323740.ch3.Jacob M., Jaros D., Rohm H. Recent advances in milk clotting enzymes. Int. J. Dairy Tech. 2011;64(1):14-33. DOI 10.1111/j.1471-0307.2010.00633.x.Jacob M., Jaros D., Rohm H. Recent advances in milk clotting enzymes. Int. J. Dairy Tech. 2011;64(1):14-33. DOI 10.1111/j.1471-0307.2010.00633.x.Jensen J.L., Molgaard A., Poulsen J.-C.N., Harboe M.K., Simonsen J.B., Lorentzen A.M., Hjernø K., Brink J.M., Qvist K.B., Larsen S. Camel and bovine chymosin: the relationship between their structures and cheese-making properties. Acta Crystallogr. D. Biol. Crystallogr. 2013;69(Pt.5):901-913. DOI 10.1107/S0907444913003260.Jensen J.L., Molgaard A., Poulsen J.-C.N., Harboe M.K., Simonsen J.B., Lorentzen A.M., Hjernø K., Brink J.M., Qvist K.B., Larsen S. Camel and bovine chymosin: the relationship between their structures and cheese-making properties. Acta Crystallogr. D. Biol. Crystallogr. 2013;69(Pt.5):901-913. DOI 10.1107/S0907444913003260.Kappeler S.R., van den Brink H.J.M., Rahbek-Nielsen H., Farah Z., Puhan Z., Hansen E.B., Johansen E. Characterization of recombinant camel chymosin reveals superior properties for the coagulation of bovine and camel milk. Biochem. Biophys. Res. Commun. 2006;342(2):647-654. DOI 10.1016/j.bbrc.2006.02.014.Kappeler S.R., van den Brink H.J.M., Rahbek-Nielsen H., Farah Z., Puhan Z., Hansen E.B., Johansen E. Characterization of recombinant camel chymosin reveals superior properties for the coagulation of bovine and camel milk. Biochem. Biophys. Res. Commun. 2006;342(2):647-654. DOI 10.1016/j.bbrc.2006.02.014.Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680-685. DOI 10.1038/227680a0.Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680-685. DOI 10.1038/227680a0.Lamichhane P., Sharma P., Kennedy D., Kelly A.L., Sheehan J.J. Microstructure and fracture properties of semi-hard cheese: differentiating the effects of primary proteolysis and calcium solubilization. Food Res. Int. 2019;125:108525. DOI 10.1016/j.foodres.2019.108525.Lamichhane P., Sharma P., Kennedy D., Kelly A.L., Sheehan J.J. Microstructure and fracture properties of semi-hard cheese: differentiating the effects of primary proteolysis and calcium solubilization. Food Res. Int. 2019;125:108525. DOI 10.1016/j.foodres.2019.108525.Lucey J.A. Formation and physical properties of milk protein gels. J. Dairy Sci. 2002;85(2):281-294. DOI 10.3168/JDS.S0022-0302(02)74078-2.Lucey J.A. Formation and physical properties of milk protein gels. J. Dairy Sci. 2002;85(2):281-294. DOI 10.3168/JDS.S0022-0302(02)74078-2.Luo F., Jiang W.H., Yang Y.X., Li J., Jiang M.F. Cloning and expression of yak active chymosin in Pichia pastoris. Asian-Australas. J. Anim. Sci. 2016;29(9):1363-1370. DOI 10.5713/AJAS.16.0038.Luo F., Jiang W.H., Yang Y.X., Li J., Jiang M.F. Cloning and expression of yak active chymosin in Pichia pastoris. Asian-Australas. J. Anim. Sci. 2016;29(9):1363-1370. DOI 10.5713/AJAS.16.0038.Mane A., McSweeney P.L.H. Proteolysis in Irish farmhouse Camembert cheese during ripening. J. Food Biochem. 2020;44(1):1-14. DOI 10.1111/jfbc.13101.Mane A., McSweeney P.L.H. Proteolysis in Irish farmhouse Camembert cheese during ripening. J. Food Biochem. 2020;44(1):1-14. DOI 10.1111/jfbc.13101.Masotti F., Hogenboom J.A., Rosi V., de Noni I., Pellegrino L. Proteolysis indices related to cheese ripening and typicalness in PDO Grana Padano cheese. Int. Dairy J. 2010;20(5):352-359. DOI 10.1016/j.idairyj.2009.11.020.Masotti F., Hogenboom J.A., Rosi V., de Noni I., Pellegrino L. Proteolysis indices related to cheese ripening and typicalness in PDO Grana Padano cheese. Int. Dairy J. 2010;20(5):352-359. DOI 10.1016/j.idairyj.2009.11.020.Moynihan A.C., Govindasamy-Lucey S., Jaeggi J.J., Johnson M.E., Lucey J.A., McSweeney P.L.H. Effect of camel chymosin on the texture, functionality, and sensory properties of low-moisture, part-skim Mozzarella cheese. J. Dairy Sci. 2014;97(1):85-96. DOI 10.3168/jds.2013-7081.Moynihan A.C., Govindasamy-Lucey S., Jaeggi J.J., Johnson M.E., Lucey J.A., McSweeney P.L.H. Effect of camel chymosin on the texture, functionality, and sensory properties of low-moisture, part-skim Mozzarella cheese. J. Dairy Sci. 2014;97(1):85-96. DOI 10.3168/jds.2013-7081.Pedersen V.B., Christensen K.A., Foltmann B. Investigations on the activation of bovine prochymosin. Eur. J. Biochem. 1979;94(2):573-580. DOI 10.1111/j.1432-1033.1979.tb12927.x.Pedersen V.B., Christensen K.A., Foltmann B. Investigations on the activation of bovine prochymosin. Eur. J. Biochem. 1979;94(2):573-580. DOI 10.1111/j.1432-1033.1979.tb12927.x.Rogelj I., Perko B., Francky A., Penca V., Pungerčar J. Recombinant lamb chymosin as an alternative coagulating enzyme in cheese production. J. Dairy Sci. 2001;84(5):1020-1026. DOI 10.3168/jds.S0022-0302(01)74561-4.Rogelj I., Perko B., Francky A., Penca V., Pungerčar J. Recombinant lamb chymosin as an alternative coagulating enzyme in cheese production. J. Dairy Sci. 2001;84(5):1020-1026. DOI 10.3168/jds.S0022-0302(01)74561-4.Sforza S., Cavatorta V., Lambertini F., Galaverna G., Dossena A., Marchelli R. Cheese peptidomics: a detailed study on the evolution of the oligopeptide fraction in Parmigiano-Reggiano cheese from curd to 24 months of aging. J. Dairy Sci. 2012;95(7):3514-3526. DOI 10.3168/jds.2011-5046.Sforza S., Cavatorta V., Lambertini F., Galaverna G., Dossena A., Marchelli R. Cheese peptidomics: a detailed study on the evolution of the oligopeptide fraction in Parmigiano-Reggiano cheese from curd to 24 months of aging. J. Dairy Sci. 2012;95(7):3514-3526. DOI 10.3168/jds.2011-5046.Singh T.K., Drake M.A., Cadwallader K.R. Flavor of Cheddar cheese: a chemical and sensory perspective. Compr. Rev. Food Sci. Food Saf. 2003;2(4):166-189. DOI 10.1111/j.1541-4337.2003.tb00021.x.Singh T.K., Drake M.A., Cadwallader K.R. Flavor of Cheddar cheese: a chemical and sensory perspective. Compr. Rev. Food Sci. Food Saf. 2003;2(4):166-189. DOI 10.1111/j.1541-4337.2003.tb00021.x.Trono D. Recombinant enzymes in the food and pharmaceutical industries. In: Singh R.S., Singhania R.R., Pandey A., Larroche C. (Eds.) Biomass, Biofuels, Biochemicals: Advances in Enzyme Technology. Elsevier, 2019;349-387. DOI 10.1016/B978-0-444-64114-4.00013-3.Trono D. Recombinant enzymes in the food and pharmaceutical industries. In: Singh R.S., Singhania R.R., Pandey A., Larroche C. (Eds.) Biomass, Biofuels, Biochemicals: Advances in Enzyme Technology. Elsevier, 2019;349-387. DOI 10.1016/B978-0-444-64114-4.00013-3.Tyagi A., Kumar A., Mohanty A.K., Kaushik J.K., Grover S., Batish V.K. Expression of buffalo chymosin in Pichia pastoris for application in mozzarella cheese. LWT – Food Sci. Technol. 2017;84:733-739. DOI 10.1016/j.lwt.2017.06.033.Tyagi A., Kumar A., Mohanty A.K., Kaushik J.K., Grover S., Batish V.K. Expression of buffalo chymosin in Pichia pastoris for application in mozzarella cheese. LWT – Food Sci. Technol. 2017;84:733-739. DOI 10.1016/j.lwt.2017.06.033.Tyagi A., Kumar A., Yadav A.K., Chandola Saklani A., Grover S., Batish V.K. Functional expression of recombinant goat chymosin in Pichia pastoris bioreactor cultures: a commercially viable alternate. LWT – Food Sci. Technol. 2016;69:217-224. DOI 10.1016/j.lwt.2016.01.015.Tyagi A., Kumar A., Yadav A.K., Chandola Saklani A., Grover S., Batish V.K. Functional expression of recombinant goat chymosin in Pichia pastoris bioreactor cultures: a commercially viable alternate. LWT – Food Sci. Technol. 2016;69:217-224. DOI 10.1016/j.lwt.2016.01.015.Vallejo J.A., Ageitos J.M., Poza M., Villa T.G. Short communication: a comparative analysis of recombinant chymosins. J. Dairy Sci. 2012;95(2):609-613. DOI 10.3168/jds.2011-4445.Vallejo J.A., Ageitos J.M., Poza M., Villa T.G. Short communication: a comparative analysis of recombinant chymosins. J. Dairy Sci. 2012;95(2):609-613. DOI 10.3168/jds.2011-4445.Vega-Hernández M.C., Gómez-Coello A., Villar J., ClaverieMartı ́ n F. Molecular cloning and expression in yeast of caprine prochymosin. J. Biotechnol. 2004;114(1-2):69-79. DOI 10.1016/j.jbiotec.2004.06.002.Vega-Hernández M.C., Gómez-Coello A., Villar J., ClaverieMartı ́ n F. Molecular cloning and expression in yeast of caprine prochymosin. J. Biotechnol. 2004;114(1-2):69-79. DOI 10.1016/j.jbiotec.2004.06.002.Wang N., Wang K.Y., Li G., Guo W., Liu D. Expression and characterization of camel chymosin in Pichia pastoris. Protein Expr. Purif. 2015;111:75-81. DOI 10.1016/j.pep.2015.03.012.Wang N., Wang K.Y., Li G., Guo W., Liu D. Expression and characterization of camel chymosin in Pichia pastoris. Protein Expr. Purif. 2015;111:75-81. DOI 10.1016/j.pep.2015.03.012.Waterhouse A., Bertoni M., Bienert S., Studer G., Tauriello G., Gumienny R., Heer F.T., de Beer T.A.P., Rempfer C., Bordoli L., Lepore R., Schwede T. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 2018;46(W1):W296-W303. DOI 10.1093/nar/gky427.Waterhouse A., Bertoni M., Bienert S., Studer G., Tauriello G., Gumienny R., Heer F.T., de Beer T.A.P., Rempfer C., Bordoli L., Lepore R., Schwede T. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 2018;46(W1):W296-W303. DOI 10.1093/nar/gky427.Wei C., Tang B., Zhang Y., Yang K. Oxidative refolding of recombinant prochymosin. Biochem. J. 1999;340(1):345-351. DOI 10.1042/0264-6021:3400345.Wei C., Tang B., Zhang Y., Yang K. Oxidative refolding of recombinant prochymosin. Biochem. J. 1999;340(1):345-351. DOI 10.1042/0264-6021:3400345.Wei C., Zhang Y., Yang K. Chaperone-mediated refolding of recombinant prochymosin. J. Protein Chem. 2000;19(6):449-456. DOI 10.1023/A:1026593113633.Wei C., Zhang Y., Yang K. Chaperone-mediated refolding of recombinant prochymosin. J. Protein Chem. 2000;19(6):449-456. DOI 10.1023/A:1026593113633.

The authors declare that there are no conflicts of interest present.