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Article
2024
Recombinant production of Paenibacillus wynnii β-galactosidase with Komagataella phaffii
Recombinant production of Paenibacillus wynnii β-galactosidase with Komagataella phaffii
Abstract (English)
The β-galactosidase from Paenibacillus wynnii (β-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low KM value) compared to industrially used β-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, β-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce β-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 β-gal-Pw in K. phaffii either extracellularly or intracellularly.ResultsFirstly, 11 different signal peptides were tested for extracellular production of β-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of β-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular β-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric β-galactosidase activity of 7537 ± 66 µkatoNPGal/Lculture was achieved by the K. phaffii 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 ± 0.05 µkatoNPGal/gDCW/h was achieved when using the GAP promoter for β-gal-Pw production compared to the AOX1 promoter. After partial purification, a β-gal-Pw enzyme preparation with a total β-galactosidase activity of 3082 ± 98 µkatoNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter).ConclusionThis study showed that the β-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular β-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.
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Microbial cell factories, 23 (2024), 263.
https://doi.org/10.1186/s12934-024-02544-5.
ISSN: 1475-2859
London : BioMed Central
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Bechtel, A., Seitl, I., Pross, E., Hetzel, F., Keutgen, M., & Fischer, L. (2024). Recombinant production of Paenibacillus wynnii β-galactosidase with Komagataella phaffii. Microbial cell factories, 23. https://doi.org/10.1186/s12934-024-02544-5
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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{Bechtel2024,
doi = {10.1186/s12934-024-02544-5},
author = {Bechtel, Anna and Seitl, Ines and Pross, Eva et al.},
title = {Recombinant production of Paenibacillus wynnii β-galactosidase with Komagataella phaffii},
journal = {Microbial Cell Factories},
year = {2024},
volume = {23},
}