Identification of rChiA DP enzyme activity in the tick-derived Bacillus IMH/B-1 strain and its enzymatic analysis

Identification of rChiA DP enzyme activity in the tick-derived Bacillus IMH/B-1 strain and its enzymatic analysis | 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 Article Identification of rChiA DP enzyme activity in the tick-derived Bacillus IMH/B-1 strain and its enzymatic analysis Fang Liu, Jile Huge, Muhammad Adeel Hassan, Ali Shah Syed Qaswar, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6122616/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Background: This study aimed to clone and express chitinase genes from Bacillus proteolyticus strains and investigate the enzymatic properties of recombinant enzymes. Materials and methods: Bacillus proteolyticus was isolated from the body of Dermacentor nuttalli and renamed IMH/B-1. Enzymes capable of degrading chitin were identified via the transparent circle method and PCR. The chitinase A (ChiA) gene was successfully cloned, and the recombinant plasmid pET28a-rChiA DP was constructed. Recombinant chitinase protein (rChiA DP) was produced through IPTG-induced expression in Escherichia coli BL21 and purified via nickel column affinity chromatography. Bioinformatic tools were used to predict the rChiA-DP protein sequences, analyse its enzyme family classification, and identify key amino acid residues involved in substrate interactions. The enzymatic activity of rChiA-DP, along with its antiparasitic and antifungal effects on Caenorhabditis elegans (C. elegans) and fungi (Aspergillus sp.), was evaluated under various conditions, such as various temperatures, pH values, metal ions, salt concentrations and substrates. Results: The amino acid sequence of the rChiA DP contains a chitin-binding domain (CBD), a fibronectin type III domain (FN3), and a catalytic domain with a typical TIM barrel molecular structure. SDS‒PAGE analysis revealed that the molecular weight of the rChiA DP was approximately 74.6 kDa, which is consistent with the theoretical predictions. The optimal conditions for rChiA-DP enzyme activity were 40°C and pH 7.0. Enzyme activity was significantly enhanced by 10 mM Ba²⁺, Tris, K⁺, and Li⁺. Organic solvents such as methanol, ethanol, isopropanol, and isoamyl alcohol (10% concentration) also increased the activity. Conversely, positive metal ions such as Cu²⁺, Ni²⁺, Fe³⁺, Zn²⁺ and Mn²⁺ as well as SDS, DMSO, Tween 80, and Tween 20 significantly inhibited the activity of the rChiA DP. rChiA DPs demonstrated varying degrees of decomposition activity against substrates such as colloidal chitin, chitin powder, nematode eggs, nematodes, shrimp shells, and tick eggs, with the highest activity observed for colloidal chitin (7.529±0.859 U/ml). However, it exhibited no activity against chitosan or ticks. Compared with the control group (high-temperature inactivation treatment) and the s-buffer group, the rChiA DP treatment significantly reduced the survival rate of C. elegans by 50.4% and 55.2%, indicating a potent antiparasitic effect (P<0.01). However, it showed no significant antifungal activity against fungi such as Aspergillus niger and Aspergillus flavus. Conclusions: This study successfully prepared tick-derived rChiA DPs and evaluated their enzymatic activity and antiparasitic effects, providing a foundation for the development of new biological pesticides. Biological sciences/Genetics Biological sciences/Microbiology Bacillus Recombinant chitinase Caenorhabditis elegans Fungi Enzymatic activity Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 01 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 24 Jun, 2025 Reviews received at journal 21 Jun, 2025 Reviews received at journal 21 Jun, 2025 Reviewers agreed at journal 16 Jun, 2025 Reviews received at journal 31 Mar, 2025 Reviewers agreed at journal 20 Mar, 2025 Reviewers agreed at journal 19 Mar, 2025 Reviewers invited by journal 18 Mar, 2025 Editor assigned by journal 18 Mar, 2025 Editor invited by journal 18 Mar, 2025 Submission checks completed at journal 14 Mar, 2025 First submitted to journal 27 Feb, 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. 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-6122616","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":431715646,"identity":"6b538298-cfa0-44cc-ad3b-d5e5244592ca","order_by":0,"name":"Fang Liu","email":"","orcid":"","institution":"Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Fang","middleName":"","lastName":"Liu","suffix":""},{"id":431715647,"identity":"e7fcfb0b-73e2-4114-b1f7-2373747eb479","order_by":1,"name":"Jile Huge","email":"","orcid":"","institution":"Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jile","middleName":"","lastName":"Huge","suffix":""},{"id":431715648,"identity":"7f243bee-a0b3-456f-b25d-da6dcaf32de8","order_by":2,"name":"Muhammad Adeel Hassan","email":"","orcid":"","institution":"Cholistan University of Veterinary and Animal Sciences Bahawalpur","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Adeel","lastName":"Hassan","suffix":""},{"id":431715649,"identity":"2139483b-fba8-4a73-a73b-78546c232a8c","order_by":3,"name":"Ali Shah Syed Qaswar","email":"","orcid":"","institution":"Cholistan University of Veterinary and Animal Sciences Bahawalpur","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"Shah Syed","lastName":"Qaswar","suffix":""},{"id":431715650,"identity":"ca6e9e01-beb5-46cc-9ce1-88e488bc1077","order_by":4,"name":"Yuxin Liu","email":"","orcid":"","institution":"Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yuxin","middleName":"","lastName":"Liu","suffix":""},{"id":431715651,"identity":"708ec520-6530-4795-8d13-aedce7d47835","order_by":5,"name":"Gele Teri","email":"","orcid":"","institution":"Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Gele","middleName":"","lastName":"Teri","suffix":""},{"id":431715652,"identity":"0d72ff13-dd8f-490b-b448-9a64b7f4371f","order_by":6,"name":"Geqi Yila","email":"","orcid":"","institution":"Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Geqi","middleName":"","lastName":"Yila","suffix":""},{"id":431715653,"identity":"9fc2c275-8a3d-4e2d-82fd-bbd157636277","order_by":7,"name":"Rina Su","email":"","orcid":"","institution":"Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Rina","middleName":"","lastName":"Su","suffix":""},{"id":431715654,"identity":"3de283e0-77f6-4741-8411-2d6b2b22a0da","order_by":8,"name":"Letu Geri","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYJCCAw/AFPOBAxB+AhFaEhgMgBRbAvFaGCBaeAzgXLxA3r334YGEij+J82fkfDx0o+YwAz97jgHDzx24tRieOW5wIOGMQeKGG7kbDuccO8wg2fPGgLH3DB4tM9IYDiS2AbVIgLSwHWYwuJFjwMzYhkfL/GdALf8MQA57cDjn32EGe0Ja5CXYgFoaDBIbbuQwHM5tA9oiQUCLAQ/QYQnHjI03nHlmcDi3L51H4syzgoO9+GxpP8b84UONnOz89uTHn3O+WcvxtydvfPATny0HYCyBBDDFAyIOYFMKt6UBxuLHq24UjIJRMApGMgAAig5bLP+Id9wAAAAASUVORK5CYII=","orcid":"","institution":"Inner Mongolia Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Letu","middleName":"","lastName":"Geri","suffix":""}],"badges":[],"createdAt":"2025-02-27 16:08:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6122616/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6122616/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-17223-7","type":"published","date":"2025-10-01T15:57:37+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":92884607,"identity":"13648e4e-cb76-46dd-9422-76a5ae3eb472","added_by":"auto","created_at":"2025-10-06 16:13:36","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3553445,"visible":true,"origin":"","legend":"","description":"","filename":"Articlemodified.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6122616/v1_covered_747edd92-cb30-4a5f-a873-56f604d1f7ce.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification of rChiA DP enzyme activity in the tick-derived Bacillus IMH/B-1 strain and its enzymatic analysis","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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bacillus, Recombinant chitinase, Caenorhabditis elegans, Fungi, Enzymatic activity","lastPublishedDoi":"10.21203/rs.3.rs-6122616/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6122616/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: This study aimed to clone and express chitinase genes from Bacillus proteolyticus strains and investigate the enzymatic properties of recombinant enzymes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMaterials and methods: Bacillus proteolyticus was isolated from the body of Dermacentor nuttalli and renamed IMH/B-1. Enzymes capable of degrading chitin were identified via the transparent circle method and PCR. The chitinase A (ChiA) gene was successfully cloned, and the recombinant plasmid pET28a-rChiA DP was constructed. Recombinant chitinase protein (rChiA DP) was produced through IPTG-induced expression in Escherichia coli BL21 and purified via nickel column affinity chromatography. Bioinformatic tools were used to predict the rChiA-DP protein sequences, analyse its enzyme family classification, and identify key amino acid residues involved in substrate interactions. The enzymatic activity of rChiA-DP, along with its antiparasitic and antifungal effects on Caenorhabditis elegans (C. elegans) and fungi (Aspergillus sp.), was evaluated under various conditions, such as various temperatures, pH values, metal ions, salt concentrations and substrates.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResults: The amino acid sequence of the rChiA DP contains a chitin-binding domain (CBD), a fibronectin type III domain (FN3), and a catalytic domain with a typical TIM barrel molecular structure. SDS‒PAGE analysis revealed that the molecular weight of the rChiA DP was approximately 74.6 kDa, which is consistent with the theoretical predictions. The optimal conditions for rChiA-DP enzyme activity were 40°C and pH 7.0. Enzyme activity was significantly enhanced by 10 mM Ba²⁺, Tris, K⁺, and Li⁺. Organic solvents such as methanol, ethanol, isopropanol, and isoamyl alcohol (10% concentration) also increased the activity. Conversely, positive metal ions such as Cu²⁺, Ni²⁺, Fe³⁺, Zn²⁺ and Mn²⁺ as well as SDS, DMSO, Tween 80, and Tween 20 significantly inhibited the activity of the rChiA DP. rChiA DPs demonstrated varying degrees of decomposition activity against substrates such as colloidal chitin, chitin powder, nematode eggs, nematodes, shrimp shells, and tick eggs, with the highest activity observed for colloidal chitin (7.529±0.859 U/ml). However, it exhibited no activity against chitosan or ticks. Compared with the control group (high-temperature inactivation treatment) and the s-buffer group, the rChiA DP treatment significantly reduced the survival rate of C. elegans by 50.4% and 55.2%, indicating a potent antiparasitic effect (P\u0026lt;0.01). However, it showed no significant antifungal activity against fungi such as Aspergillus niger and Aspergillus flavus.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConclusions: This study successfully prepared tick-derived rChiA DPs and evaluated their enzymatic activity and antiparasitic effects, providing a foundation for the development of new biological pesticides.\u003c/p\u003e","manuscriptTitle":"Identification of rChiA DP enzyme activity in the tick-derived Bacillus IMH/B-1 strain and its enzymatic analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-27 04:11:18","doi":"10.21203/rs.3.rs-6122616/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-24T17:52:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-22T00:44:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-21T21:08:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"269700037568531556299670668809120592382","date":"2025-06-16T17:05:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-31T20:23:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"44997854932293481308898968399991569048","date":"2025-03-20T17:08:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"169758193162321461240530660047017171276","date":"2025-03-19T11:27:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-18T13:10:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-18T13:07:30+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-18T04:53:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-14T16:43:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-02-27T16:02:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1cc73108-8b8a-4a52-a75f-bbbdb14709c4","owner":[],"postedDate":"March 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":45985078,"name":"Biological sciences/Genetics"},{"id":45985079,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-10-06T16:11:22+00:00","versionOfRecord":{"articleIdentity":"rs-6122616","link":"https://doi.org/10.1038/s41598-025-17223-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-10-01 15:57:37","publishedOnDateReadable":"October 1st, 2025"},"versionCreatedAt":"2025-03-27 04:11:18","video":"","vorDoi":"10.1038/s41598-025-17223-7","vorDoiUrl":"https://doi.org/10.1038/s41598-025-17223-7","workflowStages":[]},"version":"v1","identity":"rs-6122616","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6122616","identity":"rs-6122616","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}