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The current study investigates the therapeutic effect of Nattokinase, a fibrinolytic protein from the mutant strain <jats:italic>Bacillus subtilis<\/jats:italic> VITMS 2 isolated from fermented milk of <jats:italic>Vigna unguiculata<\/jats:italic>.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>The enzyme production was improved using random mutagenesis combined with statistical optimization through composite central design (CCD) with agro-residual substrates. Among all the different combinations employed, 10% <jats:italic>(v\/v)<\/jats:italic> cane molasses, 12.5\u00a0g\/L soybean waste, 12.5\u00a0g\/L eggshell powder, and 27.5\u00a0g\/L brewer\u2019s spent grain demonstrated a significant influence on fibrinolytic enzyme yield was 4639.43\u2009\u00b1\u200910.65 FU\/mL (Fibrinolytic units\/milliliter). This represents a\u2009~\u200937.75-fold increase when compared to the unoptimized wild-type strain. The CCD model demonstrated high significance (<jats:italic>p<\/jats:italic>\u2009&lt;\u20090.0001) with a strong correlation (R<jats:sup>2<\/jats:sup>\u00a0= 0.9963), indicating a reliable model fit. The identity and purity of the enzyme was confirmed via MALDI-TOF.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusion<\/jats:title>\n            <jats:p>The combination of strain improvement through mutagenesis and media optimization enhanced Nattokinase production, offering a promising approach to develop an enzyme therapy for ischemic stroke and myocardial infarction.<\/jats:p>\n          <\/jats:sec>","DOI":"10.1186\/s12866-025-04150-w","type":"journal-article","created":{"date-parts":[[2025,7,8]],"date-time":"2025-07-08T02:21:59Z","timestamp":1751941319000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Hyperproduction of nattokinase from Bacillus subtilis VIT MS2 using random mutagenesis and statistical optimization through central composite design"],"prefix":"10.1186","volume":"25","author":[{"given":"Merlyn Keziah","family":"S.","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mohanasrinivasan","family":"V.","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maneesha","family":"M.","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Subathra Devi","family":"C.","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,7,8]]},"reference":[{"issue":"S386","key":"4150_CR1","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1111\/j.0954-6820.1962.tb03046.x","volume":"172","author":"S Blix","year":"1962","unstructured":"Blix S. 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