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Plant Sci."],"abstract":"<jats:p>Nattokinase is a potent fibrinolytic enzyme widely used in the treatment of cardiovascular diseases for its ability to directly degrade fibrin and plasmin substrates, effectively dissolving blood clots. In this study, both full-length and mature forms of the nattokinase coding sequence were transiently expressed in <jats:italic>Nicotiana benthamiana<\/jats:italic> using a modified Bean Yellow Dwarf Virus (BeYDV) replicon system. Overexpression of the full-length (pre-pro) construct resulted in severe leaf necrosis within 2.5 days post-infiltration (dpi), with electrolyte leakage analysis indicating an 83.5% loss of membrane integrity by 3 dpi. In contrast, the mature form of nattokinase was successfully expressed without early cytotoxicity and exhibited strong caseinolytic and fibrinolytic activity, reaching 22,500 FU\/g\u2014comparable to commercial standards. These findings demonstrate the feasibility of producing biologically active nattokinase in plants and highlight the potential of plant-based expression systems as scalable, cost-effective platforms for therapeutic enzyme production.<\/jats:p>","DOI":"10.3389\/fpls.2025.1631697","type":"journal-article","created":{"date-parts":[[2025,7,21]],"date-time":"2025-07-21T05:22:03Z","timestamp":1753075323000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["Transient expression of full-length and mature nattokinase in Nicotiana benthamiana reveals early necrosis from full-length form and functional activity of the mature enzyme"],"prefix":"10.3389","volume":"16","author":[{"given":"Kevin","family":"Wang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hugh","family":"Mason","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kylie","family":"Hall","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ethan","family":"Slone","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kylie","family":"Tackett","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nan","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,7,21]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"773","DOI":"10.1007\/BF00020476","article-title":"Protein stability and degradation in chloroplasts","volume":"32","author":"Adam","year":"1996","journal-title":"Plant Mol. 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