Heterologous Expression and Biochemical Characterization of a Peanut Bowman-Birk Type Inhibitor

Heterologous Expression and Biochemical Characterization of a Peanut Bowman-Birk Type Inhibitor | 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 Heterologous Expression and Biochemical Characterization of a Peanut Bowman-Birk Type Inhibitor Lei Cheng, Kangming Tian, Nokuthula Peace Mchunu, Dandan Niu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-398434/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 2 You are reading this latest preprint version Abstract Objectives To heterologously express peanut ( Arachis ipaensis ) Bowman-Birk inhibitor (BBI) and characterize its properties. Results A putative BBI gene from peanut was overexpressed in Pichia pastoris with the maximal yield of 11.1 mg/L in a 250-mL shaking flask fermentation. The recombinant peanut BBI (rPBBI) was purified and its molecular weight was estimated to be 9 kDa. Purified rPBBI showed 5223.6 TIA/mg inhibitory activity toward trypsin. It retained more than 95% of its inhibitory activity over wide range of temperatures (40 to 90 o C) and pH (2.0 to 10.0) after incubation for 60 min. When presence of 100 mM dithiothreitol, rPBBI lost more than 80% inhibitory activity in 30 min. Conclusion The current investigation expressed a peanut BBI with stable inhibitory activity against trypsin and exposed the potential of heterologous overexpression approach in large scale production of BBI. Biotechnology and Bioengineering Chemical Engineering Bowman-Birk inhibitor Arachis ipaensis Overexpression Biochemical characterization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Full Text Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the manuscript can be downloaded and accessed as a PDF. Cite Share Download PDF Status: Under Revision Version 1 posted Editor assigned by journal 05 Apr, 2021 First submitted to journal 05 Apr, 2021 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-398434","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":20721753,"identity":"7ec64e48-8af8-4a51-bacb-dea1fcc7a7f8","order_by":0,"name":"Lei Cheng","email":"","orcid":"https://orcid.org/0000-0001-5654-8372","institution":"Tianjin univerisity of science and technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Cheng","suffix":""},{"id":20721754,"identity":"92ce87d0-65c9-4779-af0d-352fe98601b6","order_by":1,"name":"Kangming Tian","email":"","orcid":"","institution":"Tianjin University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kangming","middleName":"","lastName":"Tian","suffix":""},{"id":20721755,"identity":"7298957d-cddd-412a-8c8b-ec756b5afbb0","order_by":2,"name":"Nokuthula Peace Mchunu","email":"","orcid":"","institution":"Agricultural Research Council, Biotechnology Platform","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nokuthula","middleName":"Peace","lastName":"Mchunu","suffix":""},{"id":20721756,"identity":"f9012d97-7d57-4717-a64a-50dc85a8d44e","order_by":3,"name":"Dandan Niu","email":"","orcid":"","institution":"Tianjin University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dandan","middleName":"","lastName":"Niu","suffix":""},{"id":20721757,"identity":"185a120d-892d-460f-8b78-9cb9ef1cc365","order_by":4,"name":"Meng Zhang","email":"","orcid":"","institution":"Tianjin University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Meng","middleName":"","lastName":"Zhang","suffix":""},{"id":20721758,"identity":"349bced6-fecb-4c8e-82e8-ff3cf2776cb7","order_by":5,"name":"Zheng-Xiang Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYPACiXo29uYDUE4CUVpsEvh4jiU2MDAYEK0lLUFOIseQOC3yM3IMPxf8OpzHJpHz/THPnz8M/Ow5Bgw/d+DWwthzxlh6Zt/hYjaetxubedsMGCR73hgw9p7BrYWZvXeDNG/PYcY29lyglgYDBoMbOQbMjG24tbAx827+DdbCkPOwmeePAYM9IS087L3bpHl+pCW2ceQwNvOwAW2RIKBFguf8N2veBhtjNp5jhjPnthnzSJx5VnCwF48W+Rlpybd5/kjIybc3P/jw5o+cHH978sYHP/FoAQNkZ/CAiAMENADBH8JKRsEoGAWjYAQDAC0STg7JKelfAAAAAElFTkSuQmCC","orcid":"","institution":"Tianjin University of Science and Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Zheng-Xiang","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2021-04-06 16:02:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-398434/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-398434/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":8447688,"identity":"771a8dd5-a7e4-41ab-b061-63e03c81661a","added_by":"auto","created_at":"2021-04-26 14:17:51","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":97353,"visible":true,"origin":"","legend":"Amino acid sequence alignment of PBBI with five reported peanut BBIs. The sequence of signal peptides is colored in gray. The amino acids composed the reactive loop are boxed. The reactive bonds for trypsin are marked with ▲. The protease binding sites are marked with ○, while the disulfide bond related amino acids with ●. The GenBank accessory numbers for PBBI is XP_016175001, A-II, A-I, B-I and B-III is P01066 and B-II is P01067.","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-398434/v1/1865aa3e9ca02d19bea3212b.jpg"},{"id":8447685,"identity":"2d559681-2620-4786-9b03-87361f5249c6","added_by":"auto","created_at":"2021-04-26 14:17:51","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":35088,"visible":true,"origin":"","legend":"Predicted 3D structures of PBBI and A-II created by SWISS-MODEL. a: Predicted 3D structures of PBBI aligned with crystal structure of barley BBI, PDB ID: 1c2a.1.A; b: Predicted 3D structures of A-II aligned with crystal structure of chickpea BBI, PDB ID: 6lh6.1.B. The amino acid residues of the reactive sites were labeled.","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-398434/v1/d2d833fc67774366df2cc557.jpg"},{"id":8448115,"identity":"5ce00585-7566-4f37-b585-16802fc1c081","added_by":"auto","created_at":"2021-04-26 14:20:51","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":43530,"visible":true,"origin":"","legend":"Profiles of purified rPBBI and activity assay. a: Tricine-SDS-PAGE chromatogram of purified rPBBI. Lanes M: Protein molecular weight standard; 1: Supernatant of GS-PBBI; 2: Purified rPBBI; 3: supernatant of P. pastoris GS115-pPIC9k as control. b: Inhibitory activity staining of rPBBI against trypsin. c: Inhibitory activity staining of rPBBI against chymotrypsin.","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-398434/v1/39dcf61114901b3b0c537484.jpg"},{"id":8448442,"identity":"17cbeb29-4e4c-48d6-a667-5bcc53db7c43","added_by":"auto","created_at":"2021-04-26 14:23:51","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":50584,"visible":true,"origin":"","legend":"Lineweaver-Burk plots of trypsin activity-rPBBI kinetics. Trypsin activity assays were conducted with 0.4, 0.6, 0.8, and 1.0 mM of L-BAPA at 37 oC and pH 8.0, with 0 (●), 5 (▲), and 10 (■) μg/mL of rPBBI, respectively.","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-398434/v1/4c487e5db3623793e8efb615.jpg"},{"id":8448117,"identity":"7fc1899a-8685-48c4-a1bf-553d29d28d65","added_by":"auto","created_at":"2021-04-26 14:20:51","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":39535,"visible":true,"origin":"","legend":"Effect of temperature, pH, and chemicals on the trypsin inhibitory activity of rPBBI. a:\nTemperature. The thermostability of rPBBI was determined by incubating rPBBI at pH 8.2 and 40, 50, 60, 70, 80 or 90 oC for 1 h, the residual inhibitory activities were then determined according to the method part; b: pH. The rPBBI was incubated in buffers ranged from pH 2.0 to 11.0 at room temperature for 1 h, the pH was adjusted to 8.0 and the residual inhibitory activity was determined; c: DTT. The rPBBI was incubated with 1mM (▲), 10 mM (●), and 100 mM (■) of DTT for 120 min and the residue activity of rPBBI was assayed according 330 to the method part.","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-398434/v1/50fe8ab1d3e30784c7be064c.jpg"},{"id":13625054,"identity":"8f841877-ea75-4a1d-b4e9-9c8a5a4c9980","added_by":"auto","created_at":"2021-09-17 07:24:58","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":877913,"visible":true,"origin":"","legend":"","description":"","filename":"ExpressionandCharacterizationofPeanutInhibitor.pdf","url":"https://assets-eu.researchsquare.com/files/rs-398434/v1_covered.pdf"},{"id":8448718,"identity":"e7c1241f-712d-4b13-83d6-ed4b5d79b45e","added_by":"auto","created_at":"2021-04-26 14:26:56","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":641495,"visible":true,"origin":"","legend":"","description":"","filename":"ExpressionandCharacterizationofPeanutInhibitor.pdf","url":"https://assets-eu.researchsquare.com/files/rs-398434/v1_stamped.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eHeterologous Expression and Biochemical Characterization of a Peanut Bowman-Birk Type Inhibitor\u003c/p\u003e","fulltext":[{"header":"Full Text","content":"\u003cp\u003eDue to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the manuscript can be downloaded and accessed as a PDF.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biotechnology-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bile","sideBox":"Learn more about [Biotechnology Letters](https://www.springer.com/journal/10529)","snPcode":"10529","submissionUrl":"https://submission.nature.com/new-submission/10529/3","title":"Biotechnology Letters","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bowman-Birk inhibitor, Arachis ipaensis, Overexpression, Biochemical characterization","lastPublishedDoi":"10.21203/rs.3.rs-398434/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-398434/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjectives\u0026nbsp;\u003c/strong\u003eTo heterologously express peanut\u0026nbsp;( Arachis ipaensis )\u0026nbsp;Bowman-Birk inhibitor (BBI)\u0026nbsp;and characterize its properties. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u0026nbsp;\u003c/strong\u003eA putative BBI\u0026nbsp;gene from peanut\u0026nbsp;was overexpressed in Pichia pastoris with the maximal yield of 11.1\u0026nbsp;mg/L in a 250-mL shaking flask fermentation. The recombinant peanut BBI (rPBBI)\u0026nbsp;was purified and its molecular weight was estimated to be 9 kDa.\u0026nbsp;Purified rPBBI showed 5223.6 TIA/mg inhibitory activity toward trypsin. It retained more than 95% of its inhibitory activity over wide range of temperatures (40\u0026nbsp;to 90 o C) and pH (2.0\u0026nbsp;to 10.0)\u0026nbsp;after incubation for 60 min.\u0026nbsp;When presence of 100 mM dithiothreitol, rPBBI lost more than 80% inhibitory activity in 30 min.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion\u0026nbsp;\u003c/strong\u003eThe current investigation expressed a peanut BBI with stable inhibitory activity against trypsin and exposed the potential of heterologous overexpression approach in large scale production of BBI.\u003c/p\u003e","manuscriptTitle":"Heterologous Expression and Biochemical Characterization of a Peanut Bowman-Birk Type Inhibitor","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-04-26 14:17:49","doi":"10.21203/rs.3.rs-398434/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2021-04-06T00:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biotechnology Letters","date":"2021-04-05T08:45:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biotechnology-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bile","sideBox":"Learn more about [Biotechnology Letters](https://www.springer.com/journal/10529)","snPcode":"10529","submissionUrl":"https://submission.nature.com/new-submission/10529/3","title":"Biotechnology Letters","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9a31c69a-9864-4f02-a471-f54cde0f405e","owner":[],"postedDate":"April 26th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":3881724,"name":"Biotechnology and Bioengineering"},{"id":3881725,"name":"Chemical Engineering"}],"tags":[],"updatedAt":"2021-07-19T15:11:06+00:00","versionOfRecord":[],"versionCreatedAt":"2021-04-26 14:17:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-398434","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-398434","identity":"rs-398434","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}