{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,30]],"date-time":"2026-05-30T00:32:11Z","timestamp":1780101131561,"version":"3.54.0"},"reference-count":37,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2023,12,11]],"date-time":"2023-12-11T00:00:00Z","timestamp":1702252800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biotechnology Journal"],"published-print":{"date-parts":[[2024,1]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Unspecific peroxygenases (UPOs) are promising biocatalysts that catalyze oxyfunctionalization reactions without the need for costly cofactors. <jats:italic>Pichia pastoris<\/jats:italic> (reclassified as <jats:italic>Komagataella phaffii<\/jats:italic>) is considered an attractive host for heterologous expression of UPOs. However, integration of UPO\u2010expression cassettes into the genome via a single cross\u2010over yields recombinant <jats:italic>Pichia<\/jats:italic> transformants with different UPO gene copy numbers resulting in different expression levels. Selection of the most productive <jats:italic>Pichia<\/jats:italic> transformants by a commonly used screening in liquid medium in 96\u2010well plates is laborious and lasts up to 5 days. In this work, we developed a simple two\u2010step agar plate\u2010based assay to screen <jats:italic>P. pastoris<\/jats:italic> transformants for UPO activity with less effort, within shorter time, and without automated screening devices. After cell growth and protein expression on agar plates supplemented with methanol and 2,2\u2032\u2010azino\u2010bis(3\u2010ethylbenzothiazoline\u20106\u2010sulfonic acid) (ABTS), an additional top agar layer supplemented with ABTS and peroxide is added. UPO activity is visualized within 15\u00a0min by formation of green zones around UPO\u2010secreting <jats:italic>P. pastoris<\/jats:italic> transformants. The assay was validated with two UPOs, <jats:italic>Abr<\/jats:italic>UPO from <jats:italic>Aspergillus brasiliensis<\/jats:italic> and evolved PaDa\u2010I from <jats:italic>Agrocybe aegerita<\/jats:italic>. The assay results were confirmed in a quantitative 96\u2010deep well plate screening in liquid medium.<\/jats:p>","DOI":"10.1002\/biot.202300421","type":"journal-article","created":{"date-parts":[[2023,12,4]],"date-time":"2023-12-04T03:33:27Z","timestamp":1701660807000},"source":"Crossref","is-referenced-by-count":1,"title":["Agar plate\u2010based activity assay for easy and fast screening of recombinant <i>Pichia pastoris<\/i> expressing unspecific peroxygenases"],"prefix":"10.1002","volume":"19","author":[{"given":"Fabian","family":"Schmitz","sequence":"first","affiliation":[{"name":"Institute of Biochemistry Heinrich Heine University D\u00fcsseldorf, Universit\u00e4tsstra\u00dfe 1  D\u00fcsseldorf Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Annika","family":"R\u00f6der","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry Heinrich Heine University D\u00fcsseldorf, Universit\u00e4tsstra\u00dfe 1  D\u00fcsseldorf Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maike","family":"Hoffrogge","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry Heinrich Heine University D\u00fcsseldorf, Universit\u00e4tsstra\u00dfe 1  D\u00fcsseldorf Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vlada B.","family":"Urlacher","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry Heinrich Heine University D\u00fcsseldorf, Universit\u00e4tsstra\u00dfe 1  D\u00fcsseldorf Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9689-9863","authenticated-orcid":false,"given":"Katja","family":"Koschorreck","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry Heinrich Heine University D\u00fcsseldorf, Universit\u00e4tsstra\u00dfe 1  D\u00fcsseldorf Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2023,12,11]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/35051736"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cbpa.2013.03.017"},{"key":"e_1_2_10_4_1","first-page":"1","article-title":"Nature Reviews Methods Primers","author":"Bell E. L.","year":"2021","journal-title":"Biocatalysis"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.1021\/acscatal.0c01708"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-3-030-29541-7_14"},{"key":"e_1_2_10_7_1","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.70.8.4575-4581.2004"},{"key":"e_1_2_10_8_1","doi-asserted-by":"publisher","DOI":"10.1021\/acscatal.1c03065"},{"key":"e_1_2_10_9_1","doi-asserted-by":"crossref","unstructured":"Schmitz F. Koschorreck K. Hollmann F. &Urlacher V. B.(2023).Aromatic hydroxylation of substituted benzenes by an unspecific peroxygenase fromAspergillus brasiliensis.Reaction Chemistry & Engineering 8 2177\u20132186.","DOI":"10.1039\/D3RE00209H"},{"key":"e_1_2_10_10_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M010571200"},{"key":"e_1_2_10_11_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.24904"},{"key":"e_1_2_10_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.enzmictec.2015.03.004"},{"key":"e_1_2_10_13_1","doi-asserted-by":"publisher","DOI":"10.1038\/s42003-021-02076-3"},{"key":"e_1_2_10_14_1","doi-asserted-by":"publisher","DOI":"10.3390\/antiox11020223"},{"key":"e_1_2_10_15_1","unstructured":"Kimani V. W.(2019).PhD Doctoral Thesis Technical University Dresden."},{"key":"e_1_2_10_16_1","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.201600677"},{"key":"e_1_2_10_17_1","doi-asserted-by":"publisher","DOI":"10.3390\/catal13010206"},{"key":"e_1_2_10_18_1","doi-asserted-by":"publisher","DOI":"10.3390\/antiox11050891"},{"key":"e_1_2_10_19_1","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.202200558"},{"key":"e_1_2_10_20_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00253-020-10878-4"},{"key":"e_1_2_10_21_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13568-017-0368-3"},{"key":"e_1_2_10_22_1","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.201800500"},{"key":"e_1_2_10_23_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12934-016-0486-7"},{"key":"e_1_2_10_24_1","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.02712-17"},{"key":"e_1_2_10_25_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.27156"},{"key":"e_1_2_10_26_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbiotec.2022.01.006"},{"key":"e_1_2_10_27_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-0775-6_30"},{"key":"e_1_2_10_28_1","first-page":"1","article-title":"Functional expression of a blood tolerant laccase in Pichia\u2005pastoris","volume":"13","author":"Mate D. M.","year":"2013","journal-title":"BMC Biotechnology [Electronic Resource]"},{"key":"e_1_2_10_29_1","doi-asserted-by":"publisher","DOI":"10.3390\/antiox9040280"},{"key":"e_1_2_10_30_1","first-page":"4","article-title":"Cumene hydroperoxide explosion","volume":"71","author":"Francisco M.","year":"1993","journal-title":"Chemical & Engineering News"},{"key":"e_1_2_10_31_1","doi-asserted-by":"publisher","DOI":"10.1205\/095758298529623"},{"key":"e_1_2_10_32_1","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.00490-14"},{"key":"e_1_2_10_33_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi00052a031"},{"key":"e_1_2_10_34_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi960459z"},{"key":"e_1_2_10_35_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0162-0134(00)00097-0"},{"key":"e_1_2_10_36_1","doi-asserted-by":"publisher","DOI":"10.1046\/j.1432-1033.2001.01872.x"},{"key":"e_1_2_10_37_1","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.8b00500"},{"key":"e_1_2_10_38_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00253-021-11585-4"}],"container-title":["Biotechnology Journal"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/analyticalsciencejournals.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/biot.202300421","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T16:16:55Z","timestamp":1759940215000},"score":1,"resource":{"primary":{"URL":"https:\/\/analyticalsciencejournals.onlinelibrary.wiley.com\/doi\/10.1002\/biot.202300421"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,12,11]]},"references-count":37,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2024,1]]}},"alternative-id":["10.1002\/biot.202300421"],"URL":"https:\/\/doi.org\/10.1002\/biot.202300421","archive":["Portico"],"relation":{},"ISSN":["1860-6768","1860-7314"],"issn-type":[{"value":"1860-6768","type":"print"},{"value":"1860-7314","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,12,11]]},"article-number":"2300421"}}