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However, <jats:italic>\u03b2-<\/jats:italic>gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast <jats:italic>Komagataella phaffii<\/jats:italic> was chosen to investigate its feasibility to recombinantly produce <jats:italic>\u03b2-<\/jats:italic>gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the <jats:italic>\u03b2-<\/jats:italic>gal-Pw in <jats:italic>K. phaffii<\/jats:italic> either extracellularly or intracellularly.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>Firstly, 11 different signal peptides were tested for extracellular production of <jats:italic>\u03b2<\/jats:italic>-gal-Pw by <jats:italic>K. phaffii<\/jats:italic> under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of <jats:italic>\u03b2-<\/jats:italic>gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular <jats:italic>\u03b2-<\/jats:italic>gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric <jats:italic>\u03b2-<\/jats:italic>galactosidase activity of 7537\u2009\u00b1\u200966 \u00b5kat<jats:sub><jats:italic>o<\/jats:italic>NPGal<\/jats:sub>\/L<jats:sub>culture<\/jats:sub> was achieved by the <jats:italic>K. phaffii<\/jats:italic> clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14\u2009\u00b1\u20090.05 \u00b5kat<jats:sub><jats:italic>o<\/jats:italic>NPGal<\/jats:sub>\/g<jats:sub>DCW<\/jats:sub>\/h was achieved when using the GAP promoter for <jats:italic>\u03b2-<\/jats:italic>gal-Pw production compared to the AOX1 promoter. After partial purification, a <jats:italic>\u03b2-<\/jats:italic>gal-Pw enzyme preparation with a total <jats:italic>\u03b2-<\/jats:italic>galactosidase activity of 3082\u2009\u00b1\u200998 \u00b5kat<jats:sub><jats:italic>o<\/jats:italic>NPGal<\/jats:sub> was obtained from 1\u00a0L of recombinant <jats:italic>K. phaffii<\/jats:italic> culture (using AOX1 promoter).<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusion<\/jats:title>\n                <jats:p>This study showed that the <jats:italic>\u03b2-<\/jats:italic>gal-Pw was produced intracellularly by <jats:italic>K. phaffii<\/jats:italic>, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular <jats:italic>\u03b2-<\/jats:italic>gal-Pw production with <jats:italic>K. phaffii<\/jats:italic> by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.<\/jats:p>\n              <\/jats:sec>","DOI":"10.1186\/s12934-024-02544-5","type":"journal-article","created":{"date-parts":[[2024,10,5]],"date-time":"2024-10-05T02:01:52Z","timestamp":1728093712000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Recombinant production of Paenibacillus wynnii\u00a0\u03b2-galactosidase with Komagataella phaffii"],"prefix":"10.1186","volume":"23","author":[{"given":"Anna","family":"Bechtel","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ines","family":"Seitl","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Eva","family":"Pross","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Frank","family":"Hetzel","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mario","family":"Keutgen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lutz","family":"Fischer","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2024,10,5]]},"reference":[{"key":"2544_CR1","doi-asserted-by":"publisher","first-page":"530","DOI":"10.1002\/jctb.1453","volume":"81","author":"PS Panesar","year":"2006","unstructured":"Panesar PS, Panesar R, Singh RS, Kennedy JF, Kumar H. 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