Browsing by Subject "Histamine intolerance"

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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
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.
Toward food-grade production of the Glutamicibacter halophytocola diamine oxidase using Komagataella phaffii
(2025) Bechtel, Anna; Kettner, Lucas; Hessenberger, Jan; Vlassakakis, Kenny; Fischer, Lutz; Bechtel, Anna; Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Kettner, Lucas; Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Hessenberger, Jan; Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Vlassakakis, Kenny; Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Fischer, Lutz; Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany
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.