Browsing by Person "Bechtel, Anna"

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A diamine oxidase from Glutamicibacter halophytocola for the degradation of histamine and tyramine in foods
(2025) Kettner, Lucas; Freund, Alexander; Bechtel, Anna; Costa-Catala, Judit; Fischer, Lutz; Kettner, Lucas; Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599 Stuttgart, Germany; Freund, Alexander; Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599 Stuttgart, Germany; Bechtel, Anna; Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599 Stuttgart, Germany; Costa-Catala, Judit; Departament de Nutrició, Ciències de l’Alimentació i Gastronomia, Campus de l’Alimentació de Torribera, Universitat de Barcelona, Av. Prat de la Riba 171, 08921 Santa Coloma de Gramenet, Spain; Fischer, Lutz; Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599 Stuttgart, Germany
A novel diamine oxidase (DAO) was discovered in the bacterium Glutamicibacter halophytocola (DAO-GH). The gene of DAO-GH was integrated into the genome of the yeast Komagataella phaffii and recombinantly produced under control of the methanol-inducible AOX1 promoter in a bioreactor cultivation. A high DAO activity of 70.2 ± 5.2 µkat/Lculture (5.25 ± 0.22 µkat/gprotein) was yielded after 90 h of cultivation. The DAO-GH was partially purified by the polyethyleneimine precipitation of nucleic acids, fractionated ammonium sulfate precipitation and hydrophobic interaction chromatography, resulting in a specific DAO activity of 19.7 µkat/gProtein. The DAO-GH was then biochemically investigated regarding its potential for histamine and tyramine degradation in fermented foods and the human small intestine. Interestingly, the DAO-GH showed activity even at a low pH of 5 and low temperature of 6 °C. Both histamine and tyramine were effectively degraded and DAO-GH showed especially very high affinity towards tyramine (Km of 0.009 mM). The DAO-GH was shown to be capable of degrading around 20% of the initially applied histamine in tuna paste (pH 5.6) at 5 °C within 24 h and completely degraded the histamine in a simulated intestinal fluid within 1.5 h in bioconversion experiments. The DAO-GH was spray-dried for the production of a storable enzyme preparation. Only around 17% of activity were lost in this process and the DAO-GH remained stable at room temperature for at least 3 months. The discovery of this DAO with its very advantageous biochemical properties allows the preparation of histamine-reduced or -free fermented foods by a simple enzymatic treatment or the treatment of histamine intolerance symptoms as a dietary supplement or medicine.
Recombinant production of Paenibacillus wynnii β-galactosidase with Komagataella phaffii
(2024) Bechtel, Anna; Seitl, Ines; Pross, Eva; Hetzel, Frank; Keutgen, Mario; Fischer, Lutz; Bechtel, Anna; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Seitl, Ines; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Pross, Eva; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Hetzel, Frank; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Keutgen, Mario; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Fischer, Lutz; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany
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.