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Article
2025
Toward food-grade production of the Glutamicibacter halophytocola diamine oxidase using Komagataella phaffii
Toward food-grade production of the Glutamicibacter halophytocola diamine oxidase using Komagataella phaffii
Abstract (English)
The diamine oxidase from Glutamicibacter halophytocola (DAO-GH) was recombinantly produced in K. phaffii using the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter for methanol-free production. Firstly, K. phaffii clones were generated for intracellular and secretory DAO-GH production that still possessed antibiotic resistance due to the cloning procedure. For intracellular production, a maximum intracellular DAO activity of 15,404 nkat/Lculture was achieved in fed-batch bioreactor cultivations, while for secretory production, the highest extracellular DAO activity of 6,078 nkat/Lculture was achieved using the αMF signal peptide without its EAEA sequence. The intracellularly produced DAO-GH was partially purified in several purification steps with a yield of 80%, a purification factor of about 10 and specific DAO activity of 16.7 nkat/mgprotein. The secretory DAO-GH production resulted in a specific DAO activity of 15.4 nkat/mgprotein already in the cell-free culture supernatant at the end of cultivation without further purification steps. The food industry aims to avoid the use of antimicrobial resistance in enzyme production, therefore, a new cassette plasmid with self-excisable antibiotic resistance markers was constructed for secretory DAO-GH production. The antibiotic-resistance-free K. phaffii clone generated with this plasmid achieved a maximum extracellular DAO activity of 4,770 nkat/Lculture in a fed-batch bioreactor cultivation. The DAO-GH obtained in this cultivation was spray-dried, resulting in a storable powder with 23 nkat/gpowder DAO activity and a water activity value of 0.12. This study demonstrated the secretion of recombinant DAO in a microbial host such as K. phaffii for the first time and provides a strategy for generating antibiotic-resistance-free K. phaffii clones.
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AMB express, 15 (2025), 1, 177.
https://doi.org/10.1186/s13568-025-01990-7.
ISSN: 2191-0855
Berlin/Heidelberg : Springer Berlin Heidelberg
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Bechtel, A., Kettner, L., Hessenberger, J., Vlassakakis, K., & Fischer, L. (2025). Toward food-grade production of the Glutamicibacter halophytocola diamine oxidase using Komagataella phaffii. AMB express, 15(1). https://doi.org/10.1186/s13568-025-01990-7
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English
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660 Chemical engineerin
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University bibliography
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Sustainable Development Goals
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@article{Bechtel2025,
doi = {10.1186/s13568-025-01990-7},
author = {Bechtel, Anna and Kettner, Lucas and Hessenberger, Jan et al.},
title = {Toward food-grade production of the Glutamicibacter halophytocola diamine oxidase using Komagataella phaffii},
journal = {AMB Express},
year = {2025},
volume = {15},
number = {1},
}