{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,7]],"date-time":"2026-06-07T17:09:07Z","timestamp":1780852147350,"version":"3.54.1"},"reference-count":19,"publisher":"MDPI AG","issue":"22","license":[{"start":{"date-parts":[[2025,11,12]],"date-time":"2025-11-12T00:00:00Z","timestamp":1762905600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key R&D Program of China","doi-asserted-by":"crossref","award":["2023YFD1201600"],"award-info":[{"award-number":["2023YFD1201600"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Major Project for Agricultural Microbial Industry Development of Hubei Province","award":["NYWSWZX2025-2027-10"],"award-info":[{"award-number":["NYWSWZX2025-2027-10"]}]},{"name":"National GG project","award":["NK20231704"],"award-info":[{"award-number":["NK20231704"]}]},{"name":"2024 Hubei Provincial Technological Innovation Program Project","award":["2024BBB031"],"award-info":[{"award-number":["2024BBB031"]}]},{"name":"Wuhan East Lake New Technology Development Zone \u201cUnveiling the List\u201d project","award":["2022KJB001"],"award-info":[{"award-number":["2022KJB001"]}]},{"name":"Provincial-level Science and Technology Innovation Special Fund","award":["2023DJC123"],"award-info":[{"award-number":["2023DJC123"]}]},{"name":"Key Research and Development Program of Hubei Province of China","award":["2023BBB176"],"award-info":[{"award-number":["2023BBB176"]}]},{"name":"Key Research and Development Program of Hubei Province","award":["2024BBB064"],"award-info":[{"award-number":["2024BBB064"]}]},{"name":"Wuhan Knowledge Innovation Special Project \u2018Dawning Program\u2019","award":["2023020201020433"],"award-info":[{"award-number":["2023020201020433"]}]},{"name":"GG project of Hubei Province of China","award":["HBNYHXGG2023-3"],"award-info":[{"award-number":["HBNYHXGG2023-3"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Horseradish peroxidase (HRP) is a heme-containing oxidoreductase with extensive applications in biotechnology, medical diagnostics, and environmental protection. In this study, Pichia pastoris was utilized to produce HRP. Successfully, expression strains with 1\u20135 copies of HRP-C were constructed, and the strain with the highest expression level and activity of HRP-C was obtained. Different molecular chaperones (PDI1, HAC1, BIP1) were selected, and co-expression was carried out through co-induction and separate induction methods. The results showed that the yield of HRP increased approximately 1.4 times with the assistance of PDI1 and HAC1 molecular chaperones in the 3-copy Pichia pastoris expression strain, with enzyme activities increasing by 1.2-fold and 1.3-fold, respectively. High-density fermentation of the recombinant strain transformed with BDM-PDI1-HRP-C-3C was carried out in a 50 L fermenter, and after methanol induction for 72 h, a target protein expression level of up to 200 mg\/L was achieved. The enzyme activity reached 1796 U\/mL, which is nearly three times higher than that of shake-flask fermentation and is the highest reported in the literature to date.<\/jats:p>","DOI":"10.3390\/molecules30224374","type":"journal-article","created":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T09:59:09Z","timestamp":1763027949000},"page":"4374","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Study on the High-Efficiency Expression of Horseradish Peroxidase in Pichia pastoris"],"prefix":"10.3390","volume":"30","author":[{"given":"Yaping","family":"Wang","sequence":"first","affiliation":[{"name":"National Biopesticide Engineering Technology Research Center, Hubei Biopesticide Engineering Research Center, Hubei Academy of Agricultural Sciences, Biopesticide Branch of Hubei Innovation Centre of Agricultural Science and Technology, Wuhan 430064, China"},{"name":"State Key Laboratory of Biocatalysis and Enzyme, Engineering Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei University, Wuhan 430062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yidan","family":"Jing","sequence":"additional","affiliation":[{"name":"National Biopesticide Engineering Technology Research Center, Hubei Biopesticide Engineering Research Center, Hubei Academy of Agricultural Sciences, Biopesticide Branch of Hubei Innovation Centre of Agricultural Science and Technology, Wuhan 430064, China"},{"name":"State Key Laboratory of Biocatalysis and Enzyme, Engineering Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei University, Wuhan 430062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weizhen","family":"Li","sequence":"additional","affiliation":[{"name":"National Biopesticide Engineering Technology Research Center, Hubei Biopesticide Engineering Research Center, Hubei Academy of Agricultural Sciences, Biopesticide Branch of Hubei Innovation Centre of Agricultural Science and Technology, Wuhan 430064, China"},{"name":"State Key Laboratory of Biocatalysis and Enzyme, Engineering Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei University, Wuhan 430062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuqing","family":"Wang","sequence":"additional","affiliation":[{"name":"National Biopesticide Engineering Technology Research Center, Hubei Biopesticide Engineering Research Center, Hubei Academy of Agricultural Sciences, Biopesticide Branch of Hubei Innovation Centre of Agricultural Science and Technology, Wuhan 430064, China"},{"name":"State Key Laboratory of Biocatalysis and Enzyme, Engineering Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei University, Wuhan 430062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fei","family":"Li","sequence":"additional","affiliation":[{"name":"National Biopesticide Engineering Technology Research Center, Hubei Biopesticide Engineering Research Center, Hubei Academy of Agricultural Sciences, Biopesticide Branch of Hubei Innovation Centre of Agricultural Science and Technology, Wuhan 430064, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yimin","family":"Qiu","sequence":"additional","affiliation":[{"name":"National Biopesticide Engineering Technology Research Center, Hubei Biopesticide Engineering Research Center, Hubei Academy of Agricultural Sciences, Biopesticide Branch of Hubei Innovation Centre of Agricultural Science and Technology, Wuhan 430064, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ben","family":"Rao","sequence":"additional","affiliation":[{"name":"National Biopesticide Engineering Technology Research Center, Hubei Biopesticide Engineering Research Center, Hubei Academy of Agricultural Sciences, Biopesticide Branch of Hubei Innovation Centre of Agricultural Science and Technology, Wuhan 430064, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"249","DOI":"10.1016\/j.phytochem.2003.10.022","article-title":"Horseradish peroxidase: A modern view of a classic 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Immunol. Methods"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1611","DOI":"10.1007\/s00253-014-6346-7","article-title":"An updated view on horseradish peroxidases: Recombinant production and biotechnological applications","volume":"99","author":"Krainer","year":"2015","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Mirkovi\u0107, N., Zihrul, M., \u017di\u017ei\u0107, M., Marinkovi\u0107, A., Prodanovi\u0107, R., and Todorovi\u0107, T.R. (2022). The Influence of Isoenzyme Composition and Chemical Modification on Horseradish Peroxidase@ZIF-8 Biocomposite Performance. Polymers, 14.","DOI":"10.3390\/polym14224834"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1016\/0003-9861(92)90641-9","article-title":"Baculovirus expression and characterization of catalytically active horseradish peroxidase","volume":"297","author":"Hartmann","year":"1992","journal-title":"Arch. Biochem. 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