A New Phospholipase D-Producing Bacillus cereus: Taxonomy, Mutagenesis, Fermentation Optimization and Enzyme Characterization

A New Phospholipase D-Producing Bacillus cereus: Taxonomy, Mutagenesis, Fermentation Optimization and Enzyme Characterization | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A New Phospholipase D-Producing Bacillus cereus: Taxonomy, Mutagenesis, Fermentation Optimization and Enzyme Characterization Ying He, Ao Huang, Yun Liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6034154/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 May, 2025 Read the published version in Applied Biochemistry and Biotechnology → Version 1 posted 4 You are reading this latest preprint version Abstract Phospholipase D (PLD) is a valuable enzyme in industrial processes for converting phosphatidylcholine (PC) to phosphatidylserine (PS). In this study, a strain of Bacillus cereus was isolated from soil and identified through 16S rRNA sequencing. To enhance PLD activity, various mutagenesis strategies—including chemical treatment, irradiation, and their combinations—were employed, resulting in four high-activity positive mutants (C-7, I-12, CI 7–12, and IC 13–14). Among these, the CI 7–12 mutant exhibited a significantly higher enzymatic activity, showing a 3.12-fold increase (312.2%) compared to the wild-type strain. Fermentation conditions were optimied using response surface methodology (RSM), achieving a PLD activity of 35 U/mL. The enzyme demonstrated stability over a wide temperature range (30–60°C) and pH range (6–10), with a half-life of 128 days. Kinetic analysis revealed a V max of 20.04 µmol/h and a K m of 7.13 µmol/mL, indicating efficient activity. In bioconversion experiments, the PLD-enriched fermentation broth catalyzed the conversion of PC to PS, achieving a 53.0% conversion rate and a 92.3% selectivity for PS in a two-phase system. These findings expand the potential sources of PLD and underscore its applicability for PS production in biotechnological applications. Phospholipase D (PLD) Strain taxonomy Mutagenesis Fermentation optimization Enzyme characterization Full Text Supplementary Files renamed8bcd9.docx Cite Share Download PDF Status: Published Journal Publication published 20 May, 2025 Read the published version in Applied Biochemistry and Biotechnology → Version 1 posted Editorial decision: Accept without change 02 May, 2025 Editor assigned by journal 22 Apr, 2025 Editor invited by journal 06 Apr, 2025 First submitted to journal 04 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6034154","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":444464785,"identity":"85f69e6f-2956-402a-bf5a-8fc917f5572f","order_by":0,"name":"Ying He","email":"","orcid":"","institution":"Beijing University of Chemical Technology","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"He","suffix":""},{"id":444464786,"identity":"cf26a711-1fc7-46f3-b421-064b94fd676c","order_by":1,"name":"Ao Huang","email":"","orcid":"","institution":"Beijing University of Chemical Technology","correspondingAuthor":false,"prefix":"","firstName":"Ao","middleName":"","lastName":"Huang","suffix":""},{"id":444464787,"identity":"2a1f2c03-4ee4-4881-8d4c-ee0ddbafd81d","order_by":2,"name":"Yun Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYBACPmYogw1MVjDwENTChqSFsYHhDDFakNiMDYxtRDiMjZ3HTJp3x518Ponk5w8+zquTMWc/wPjhYw4+h4G0nHlm2SaRZtg4c9thHsueBGbJmdsIaWk7bMDGc8CwmXfbAR6DAwlszLzEaTn+sfnvnDoeg/MPiNXC3mPYzNjAzGNwg6AtbMWWc9uegbQUzuw5dhio5WEzXr/w8x/eeONt2x0D+Wb2DR9+1NTZG5xPPvjhIx4tQMAiwcBwAFkAGKUEAPMHNC2jYBSMglEwClABAFd8R+3+kJGKAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-7521-3831","institution":"Beijing University of Chemical Technology","correspondingAuthor":true,"prefix":"","firstName":"Yun","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-02-15 04:03:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6034154/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6034154/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12010-025-05278-1","type":"published","date":"2025-05-20T15:58:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83460065,"identity":"ab8cfeec-476a-4380-8ca4-0a071620639e","added_by":"auto","created_at":"2025-05-26 16:09:45","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1295222,"visible":true,"origin":"","legend":"","description":"","filename":"manuscriptR1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6034154/v1_covered_9b0608ef-6e0e-499b-9bcd-5770a2c41f82.pdf"},{"id":80885015,"identity":"9d024a55-4900-4cd6-bf9e-0a8da646ef56","added_by":"auto","created_at":"2025-04-18 08:36:11","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1164900,"visible":true,"origin":"","legend":"","description":"","filename":"renamed8bcd9.docx","url":"https://assets-eu.researchsquare.com/files/rs-6034154/v1/ddc4e523e32ad76d2a8e1d82.docx"}],"financialInterests":"","formattedTitle":"A New Phospholipase D-Producing Bacillus cereus: Taxonomy, Mutagenesis, Fermentation Optimization and Enzyme Characterization","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"applied-biochemistry-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"abab","sideBox":"Learn more about [Applied Biochemistry and Biotechnology](https://www.springer.com/journal/12010)","snPcode":"12010","submissionUrl":"https://submission.nature.com/new-submission/12010/3","title":"Applied Biochemistry and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Phospholipase D (PLD), Strain taxonomy, Mutagenesis, Fermentation optimization, Enzyme characterization","lastPublishedDoi":"10.21203/rs.3.rs-6034154/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6034154/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhospholipase D (PLD) is a valuable enzyme in industrial processes for converting phosphatidylcholine (PC) to phosphatidylserine (PS). In this study, a strain of \u003cem\u003eBacillus cereus\u003c/em\u003e was isolated from soil and identified through 16S rRNA sequencing. To enhance PLD activity, various mutagenesis strategies\u0026mdash;including chemical treatment, irradiation, and their combinations\u0026mdash;were employed, resulting in four high-activity positive mutants (C-7, I-12, CI 7\u0026ndash;12, and IC 13\u0026ndash;14). Among these, the CI 7\u0026ndash;12 mutant exhibited a significantly higher enzymatic activity, showing a 3.12-fold increase (312.2%) compared to the wild-type strain. Fermentation conditions were optimied using response surface methodology (RSM), achieving a PLD activity of 35 U/mL. The enzyme demonstrated stability over a wide temperature range (30\u0026ndash;60\u0026deg;C) and pH range (6\u0026ndash;10), with a half-life of 128 days. 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