Browsing by Person "Lutz-Wahl, Sabine"

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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.
Multi-Enzymsystem zur Herstellung eines alternativen Zuckersirups
(2020) Lutz-Wahl, Sabine; Pross, Eva; Hinrichs, Jörg; Fischer, Lutz
Energy-rich foods lead to an increase of overweight people in our societies. Mainly the fat and sugar content in foods is responsible for this progress. However, both components are very important for the taste and acceptance of foods. Thus, the abundant available lactose will be enzymatically modified by using the enzymes β-galactosidase, L-arabinose isomerase and glucose isomerase in order to generate a new natural sugar syrup. The new sugar mixture will be much sweeter than lactose.
Recombinant production of acidophilic L-arabinose isomerase from Lentilactobacillus parakefiri in Bacillus subtilis
(2025) Weber, Nathanael; Götz, Sebastian; Senger, Jana; Lutz-Wahl, Sabine; Fischer, Lutz; Weber, Nathanael; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Götz, Sebastian; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Senger, Jana; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Lutz-Wahl, Sabine; 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
Background: The monosaccharide D-tagatose is a promising alternative to sucrose because of its similar sweetness and lower glycemic index. A novel L-arabinose isomerase (L-AI) from Lentilactobacillus parakefiri DSM 10551 (L-AI-Lp) has been biochemically characterized and used to isomerize D-galactose to D-tagatose in skim milk ultrafiltration permeate at pH 4.5 and 6.5. However, like most L-AIs described in the literature, this enzyme has only been produced recombinantly in Escherichia coli. This study aimed to systematically investigate the intracellular recombinant production of L-AI-Lp in Bacillus subtilis, which has qualified for a presumption of safety (QPS) designation from the European Food Safety Authority. Results: The influence of four promoters on L-AI-Lp production in B. subtilis 007 was investigated in shake flask cultivations. Among these, the PAprE promoter yielded the highest volumetric L-AI activity of 69.2 ± 7.4 µkatGal, 65 °C/LCulture. The production yield was further increased to 147.7 ± 1.0 µkatGal, 65 °C/LCulture by using the nonsporulating, surfactin-deficient strain B. subtilis 007 ∆sfp ∆sigF, which was constructed by deleting sigF and sfp in B. subtilis 007. Furthermore, the influence of pH and dissolved oxygen (DO) on bioreactor cultivations of B. subtilis 007 ∆sfp ∆sigF was analyzed. In bioreactor cultivations, the highest L-AI activity of 88.6 ± 2.4 µkatGal, 65 °C/LCulture was measured under unregulated pH and low oxygen conditions (DO ≤ 5%), representing a 3.2-fold increase compared with previous recombinant production in E. coli. The L-AI-Lp was subsequently partially purified by heat treatment and precipitation methods, resulting in a 7.8-fold increase in specific activity to 128.2 nkatGal, 65 °C/mg and a yield of 84%. Conclusions: The L-AI-Lp was recombinantly produced for the first time in a microbial species with QPS status using the nonsporulating and surfactin-deficient strain B. subtilis 007 ∆sfp ∆sigF. The L-AI-Lp was subsequently partially purified via nonchromatographic methods, providing a basis for a low-cost downstream process. These results represent an important step toward potential industrial application of L-AI-Lp and highlight the potential of B. subtilis 007 ∆sfp ∆sigF as an expression host for the recombinant production of L-AIs compared with previously used hosts from the order Lactobacillales.