Tobacco BY2 cells expressing recombinant cardosin B as an alternative for production of active milk clotting enzymes

Abstract Cynara cardunculus L. or cardoon is a plant that is used as a source of milk clotting enzymes during traditional cheese manufacturing. This clotting activity is due to aspartic proteases (APs) found in the cardoon flower, named cyprosins and cardosins. APs from cardoon flowers display a gre...

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Autores principales:	André Folgado, Rita Abranches
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b98b190139c840cd84795b0a62360d07
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spelling	oai:doaj.org-article:b98b190139c840cd84795b0a62360d072021-12-02T16:14:08ZTobacco BY2 cells expressing recombinant cardosin B as an alternative for production of active milk clotting enzymes10.1038/s41598-021-93882-62045-2322https://doaj.org/article/b98b190139c840cd84795b0a62360d072021-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-93882-6https://doaj.org/toc/2045-2322Abstract Cynara cardunculus L. or cardoon is a plant that is used as a source of milk clotting enzymes during traditional cheese manufacturing. This clotting activity is due to aspartic proteases (APs) found in the cardoon flower, named cyprosins and cardosins. APs from cardoon flowers display a great degree of heterogeneity, resulting in variable milk clotting activities and directly influencing the final product. Producing these APs using alternative platforms such as bacteria or yeast has proven challenging, which is hampering their implementation on an industrial scale. We have developed tobacco BY2 cell lines as an alternative plant-based platform for the production of cardosin B. These cultures successfully produced active cardosin B and a purification pipeline was developed to obtain isolated cardosin B. The enzyme displayed proteolytic activity towards milk caseins and milk clotting activity under standard cheese manufacturing conditions. We also identified an unprocessed form of cardosin B and further investigated its activation process. The use of protease-specific inhibitors suggested a possible role for a cysteine protease in cardosin B processing. Mass spectrometry analysis identified three cysteine proteases containing a granulin-domain as candidates for cardosin B processing. These findings suggest an interaction between these two groups of proteases and contribute to an understanding of the mechanisms behind the regulation and processing of plant APs. This work also paves the way for the use of tobacco BY2 cells as an alternative production system for active cardosins and represents an important advancement towards the industrial production of cardoon APs.André FolgadoRita AbranchesNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-11 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q André Folgado Rita Abranches Tobacco BY2 cells expressing recombinant cardosin B as an alternative for production of active milk clotting enzymes
description	Abstract Cynara cardunculus L. or cardoon is a plant that is used as a source of milk clotting enzymes during traditional cheese manufacturing. This clotting activity is due to aspartic proteases (APs) found in the cardoon flower, named cyprosins and cardosins. APs from cardoon flowers display a great degree of heterogeneity, resulting in variable milk clotting activities and directly influencing the final product. Producing these APs using alternative platforms such as bacteria or yeast has proven challenging, which is hampering their implementation on an industrial scale. We have developed tobacco BY2 cell lines as an alternative plant-based platform for the production of cardosin B. These cultures successfully produced active cardosin B and a purification pipeline was developed to obtain isolated cardosin B. The enzyme displayed proteolytic activity towards milk caseins and milk clotting activity under standard cheese manufacturing conditions. We also identified an unprocessed form of cardosin B and further investigated its activation process. The use of protease-specific inhibitors suggested a possible role for a cysteine protease in cardosin B processing. Mass spectrometry analysis identified three cysteine proteases containing a granulin-domain as candidates for cardosin B processing. These findings suggest an interaction between these two groups of proteases and contribute to an understanding of the mechanisms behind the regulation and processing of plant APs. This work also paves the way for the use of tobacco BY2 cells as an alternative production system for active cardosins and represents an important advancement towards the industrial production of cardoon APs.
format	article
author	André Folgado Rita Abranches
author_facet	André Folgado Rita Abranches
author_sort	André Folgado
title	Tobacco BY2 cells expressing recombinant cardosin B as an alternative for production of active milk clotting enzymes
title_short	Tobacco BY2 cells expressing recombinant cardosin B as an alternative for production of active milk clotting enzymes
title_full	Tobacco BY2 cells expressing recombinant cardosin B as an alternative for production of active milk clotting enzymes
title_fullStr	Tobacco BY2 cells expressing recombinant cardosin B as an alternative for production of active milk clotting enzymes
title_full_unstemmed	Tobacco BY2 cells expressing recombinant cardosin B as an alternative for production of active milk clotting enzymes
title_sort	tobacco by2 cells expressing recombinant cardosin b as an alternative for production of active milk clotting enzymes
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/b98b190139c840cd84795b0a62360d07
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Tobacco BY2 cells expressing recombinant cardosin B as an alternative for production of active milk clotting enzymes

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