Browsing by Person "Kettner, Lucas"

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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.
An extremely sensitive amylase activity assay and its application for the determination of the residual amylase activity in bread
(2025) Reichenberger, Katrin; Lutz-Wahl, Sabine; Kettner, Lucas; Fischer, Lutz
An extremely sensitive amylase activity assay was developed using the natural substrate starch and two ancillary enzymes: a glucose oxidase (GOD) and a peroxidase, to measure the residual activity of the α -amylase from Bacillus subtilis in white bread. Firstly, the concentrations of the assay components: electron acceptor DA-67 (50 μM), horseradish peroxidase (681 nkat mL −1 ), a GOD from Aspergillus niger (1550 nkat mL −1 ) and the natural substrate starch (0.01% (w/v)), were optimized to achieve high sensitivity. The linearity of the assay was then tested with both an endo- ( α -amylase from B. subtilis ) and exo-acting amylase (maltogenic amylase from Geobacillus stearothermophilus ), and the effect of the incubation time on the assay sensitivity was investigated and optimized. The optimized assay was capable of determining a minimal amylase activity of 0.33 pkat mL −1 for both amylases tested with an assay run time of 7.5 h. This new DA-67 amylase assay demonstrated 4.7- and 4.2-times higher sensitivity, respectively, compared to optimized versions of the commercial Ceralpha (determination of endo-amylase activities) and Betamyl3 (determination of exo-amylase activities) assays. The new DA-67 amylase assay was used to determine the residual activity of α -amylase from B. subtilis in white bread. A consistent residual activity of 2.26 ± 0.15% was reliably determined.