The extraction of polyphenol oxidase and the enzymatic synthesis of theaflavin are based on a three-phase separation technolog

The extraction of polyphenol oxidase and the enzymatic synthesis of theaflavin are based on a three-phase separation technolog | 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 The extraction of polyphenol oxidase and the enzymatic synthesis of theaflavin are based on a three-phase separation technolog Shiqi Zhang, Jin Lei, yu Lei, Yuhao Li, Dong Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7917504/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Polyphenol oxidase (PPO) plays a pivotal role in the conversion of catechins into theaflavins (TFs). The yield and purity of TFs produced by purified PPO are superior to those produced by crude enzymes in the enzymatic preparation of TFs. The present study utilizes a potato PPO enzyme as a donor to investigate the optimal purification conditions for three-phase separation, the enzymatic properties of the purified PPO, and its performance in the enzymatic synthesis of theaflavins. The optimal three-phase separation conditions were determined to be as follows: 50% ammonium sulfate saturation, a 1:1.5 extraction solution-to-tert-butanol volume ratio, a system pH of 5, and a 60-minute separation time. Consequently, the PPO enzyme activity attained a value of 251.00 U/mL, exhibiting a purification fold of 5.73. Furthermore, the study yielded preliminary findings regarding the enzymatic properties of the purified PPO and its application in the enzymatic synthesis of TFs. The results demonstrated that the purified enzyme exhibited a higher TF synthesis yield in comparison to the crude enzyme. In addition, the investigation revealed that potato PPO exhibited specificity toward the formation of theaflavin monomers, preferentially generating TFDG while demonstrating weaker synthesis capacity for TF-3'-G. The present study employed a three-phase separation method for potato PPO, with a short purification cycle and high extraction efficiency, advantageous for industrial TFs production. Potato Polyphenol Oxidase Theaflavin Three-Phase Separation Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7917504","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":542488646,"identity":"7f127a08-5c02-4f1b-ac0f-44ad805fe70c","order_by":0,"name":"Shiqi Zhang","email":"","orcid":"","institution":"Sichuan University of Science and Engineering","correspondingAuthor":false,"prefix":"","firstName":"Shiqi","middleName":"","lastName":"Zhang","suffix":""},{"id":542488647,"identity":"7431421a-ed50-4ef9-8beb-1fa4f5ec9b6f","order_by":1,"name":"Jin Lei","email":"","orcid":"","institution":"Sichuan University of Science and 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three-phase separation technolog","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Potato, Polyphenol Oxidase, Theaflavin, Three-Phase Separation","lastPublishedDoi":"10.21203/rs.3.rs-7917504/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7917504/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePolyphenol oxidase (PPO) plays a pivotal role in the conversion of catechins into theaflavins (TFs). The yield and purity of TFs produced by purified PPO are superior to those produced by crude enzymes in the enzymatic preparation of TFs. The present study utilizes a potato PPO enzyme as a donor to investigate the optimal purification conditions for three-phase separation, the enzymatic properties of the purified PPO, and its performance in the enzymatic synthesis of theaflavins. The optimal three-phase separation conditions were determined to be as follows: 50% ammonium sulfate saturation, a 1:1.5 extraction solution-to-tert-butanol volume ratio, a system pH of 5, and a 60-minute separation time. Consequently, the PPO enzyme activity attained a value of 251.00 U/mL, exhibiting a purification fold of 5.73. Furthermore, the study yielded preliminary findings regarding the enzymatic properties of the purified PPO and its application in the enzymatic synthesis of TFs. The results demonstrated that the purified enzyme exhibited a higher TF synthesis yield in comparison to the crude enzyme. In addition, the investigation revealed that potato PPO exhibited specificity toward the formation of theaflavin monomers, preferentially generating TFDG while demonstrating weaker synthesis capacity for TF-3'-G. The present study employed a three-phase separation method for potato PPO, with a short purification cycle and high extraction efficiency, advantageous for industrial TFs production.\u003c/p\u003e","manuscriptTitle":"The extraction of polyphenol oxidase and the enzymatic synthesis of theaflavin are based on a three-phase separation technolog","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-11 23:19:00","doi":"10.21203/rs.3.rs-7917504/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f7fc99b4-991d-4989-bd38-18354251ffa8","owner":[],"postedDate":"November 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-30T10:09:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-11 23:19:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7917504","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7917504","identity":"rs-7917504","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}