{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T11:03:03Z","timestamp":1782817383559,"version":"3.54.5"},"reference-count":59,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2024,12,11]],"date-time":"2024-12-11T00:00:00Z","timestamp":1733875200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Research Council of Thailand (NRCT)","award":["NRCT5-RRI63004-P03"],"award-info":[{"award-number":["NRCT5-RRI63004-P03"]}]},{"name":"Fundamental Fund of Khon Kean University (KKU)","award":["NRCT5-RRI63004-P03"],"award-info":[{"award-number":["NRCT5-RRI63004-P03"]}]},{"name":"Basic Research Fund of Khon Kaen University","award":["NRCT5-RRI63004-P03"],"award-info":[{"award-number":["NRCT5-RRI63004-P03"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["JoF"],"abstract":"<jats:p>Hyaluronidases have been a subject of great interest in medical and cosmeceutical applications. Previously, our group demonstrated that the venom glands of Vespa tropica contain hyaluronidase enzymes (VesT2s), and heterologous expression of the corresponding gene (VesT2a) in E. coli systems results in inclusion bodies, necessitating functional folding using urea. Here, we report the successful heterologous expression of VesT2a in the Pichia pastoris expression system, with gene construction achieved using GoldenPiCS. After confirming gene integration in the yeast genome, methanol-induced cultures yielded an exceptional amount of VesT2a, approximately two-fold higher than that obtained with the constitutive expression vector (PGAP). Upon culturing in a bioreactor, yeast cells harboring pAOX1-\u03b1MF-VesT2a produced secreted proteins with a total yield of 96.45 mg\/L. The secreted VesT2a has a molecular weight of 59.35 kDa, significantly higher than the expected molecular weight (~40.05 kDa), presumably due to endogenous glycosylation by the yeast cells. It exhibits optimal activity at 37 \u00b0C and pH 3, showing a specific activity of 4238.37 U\/mg, and remains active across a broad range of pH and temperature. Notably, it exhibits higher hyaluronidase activity than the crude venom and E. coli-expressed protein, likely due to improved folding via endogenous post-translational modifications, such as disulfide bonds and N-glycosylation; this underscores the potential of heterologous systems for producing venomous hyaluronidases from other species. In silico docking-based analyses further support its catalytic activity and provide insights into seeking natural inhibitors from phenolic-rich plant extracts to alleviate symptoms in patients suffering from insect bites and stings.<\/jats:p>","DOI":"10.3390\/jof10120854","type":"journal-article","created":{"date-parts":[[2024,12,11]],"date-time":"2024-12-11T03:14:58Z","timestamp":1733886898000},"page":"854","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Production of Vespa tropica Hyaluronidase by Pichia pastoris"],"prefix":"10.3390","volume":"10","author":[{"given":"Piyapon","family":"Janpan","sequence":"first","affiliation":[{"name":"Division of Pharmacognosy and Toxicology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand"},{"name":"Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen 40000, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bernhard","family":"Schmelzer","sequence":"additional","affiliation":[{"name":"Institute of Microbiology and Microbial Biotechnology, Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna (BOKU), 1190 Vienna, Austria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anuwatchakij","family":"Klamrak","sequence":"additional","affiliation":[{"name":"Division of Pharmacognosy and Toxicology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand"},{"name":"Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen 40000, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patthana","family":"Tastub","sequence":"additional","affiliation":[{"name":"Betagro Science Center Co., Ltd., 136 Khlong Nueng, Khlong Luang District, Pathum Thani 12120, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tewa","family":"Upathanpreecha","sequence":"additional","affiliation":[{"name":"Betagro Science Center Co., Ltd., 136 Khlong Nueng, Khlong Luang District, Pathum Thani 12120, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shaikh Shahinur","family":"Rahman","sequence":"additional","affiliation":[{"name":"Division of Pharmacognosy and Toxicology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand"},{"name":"Department of Applied Nutrition and Food Technology, Faculty of Biological Sciences, Islamic University, Kushtia 7000, Bangladesh"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jaran","family":"Nabnueangsap","sequence":"additional","affiliation":[{"name":"Salaya Central Instrument Facility RSPG, Research Management and Development Division, Office of the President, Mahidol University, Nakhon Pathom 73170, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yutthakan","family":"Saengkun","sequence":"additional","affiliation":[{"name":"Division of Pharmacognosy and Toxicology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand"},{"name":"Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen 40000, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Prapenpuksiri","family":"Rungsa","sequence":"additional","affiliation":[{"name":"Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen 40000, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Diethard","family":"Mattanovich","sequence":"additional","affiliation":[{"name":"Institute of Microbiology and Microbial Biotechnology, Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna (BOKU), 1190 Vienna, Austria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2672-7710","authenticated-orcid":false,"given":"Sakda","family":"Daduang","sequence":"additional","affiliation":[{"name":"Division of Pharmacognosy and Toxicology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand"},{"name":"Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen 40000, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,12,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1921","DOI":"10.1016\/j.lfs.2007.02.037","article-title":"The Magic Glue Hyaluronan and Its Eraser Hyaluronidase: A Biological Overview","volume":"80","author":"Girish","year":"2007","journal-title":"Life 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