scholarly journals Determination and characterisation of milk-clotting activity of two Solanum tuberosum aspartic proteases (StAPs)

2020 ◽  
Vol 104 ◽  
pp. 104645 ◽  
Author(s):  
Florencia Rocío Tito ◽  
Alfonso Pepe ◽  
Claudia Virginia Tonon ◽  
Gustavo Raúl Daleo ◽  
María Gabriela Guevara
Keyword(s):  
Solanum Tuberosum ◽  
Aspartic Proteases ◽  
Milk Clotting ◽  
Milk Clotting Activity

scholarly journals Expression of recombinant cardosin B in tobacco BY2 cells: an alternative system for the production of active milk clotting enzymes

2021 ◽  
Author(s):  
André Folgado ◽  
Rita Abranches
Keyword(s):  
Cysteine Proteases ◽  
Mass Spectrometry Analysis ◽  
Activation Process ◽  
Cynara Cardunculus ◽  
Aspartic Proteases ◽  
Spectrometry Analysis ◽  
Milk Clotting ◽  
Traditional Cheese ◽  
Milk Clotting Activity ◽  
Degree Of Heterogeneity

AbstractCynara 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.

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

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
André Folgado ◽  
Rita Abranches
Keyword(s):  
Cysteine Proteases ◽  
Mass Spectrometry Analysis ◽  
Activation Process ◽  
Cynara Cardunculus ◽  
Aspartic Proteases ◽  
Spectrometry Analysis ◽  
Milk Clotting ◽  
Traditional Cheese ◽  
Milk Clotting Activity ◽  
Degree Of Heterogeneity

AbstractCynara 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.

A novel milk-clotting enzyme from Aspergillus oryzae and A. luchuensis is an aspartic endopeptidase PepE presumed to be a vacuolar enzyme

2022 ◽  
Author(s):  
Yoko Takyu ◽  
Taro Asamura ◽  
Ayako Okamoto ◽  
Hiroshi Maeda ◽  
Michio Takeuchi ◽  
...  
Keyword(s):  
Aspergillus Oryzae ◽  
Milk Clotting ◽  
Milk Clotting Enzyme ◽  
Proteolytic Activities ◽  
Homologous Enzyme ◽  
Traditional Fermentation ◽  
Enzyme Source ◽  
Cheese Production ◽  
Milk Clotting Activity ◽  
Aspartic Endopeptidase

Abstract Aspergillus oryzae RIB40 has 11 aspartic endopeptidase genes. We searched for milk-clotting enzymes based on the homology of the deduced amino acid sequence with chymosins. As a result, we identified a milk-clotting enzyme in A. oryzae. We expected other Aspergillus species to have a homologous enzyme with milk-clotting activity, and we found the most homologous aspartic endopeptidase from A. luchuensis had milk-clotting activity. Surprisingly, two enzymes were considered as vacuole enzymes according to a study on A. niger proteases. The two enzymes from A. oryzae and A. luchuensis cleaved a peptide between the 105Phe-106Met bond in κ-casein, similar to chymosin. Although both enzymes showed proteolytic activity using casein as a substrate, the optimum pH values for milk-clotting and proteolytic activities were different. Furthermore, the substrate specificities were highly restricted. Therefore, we expected that the Japanese traditional fermentation agent, koji, could be used as an enzyme source for cheese production.

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