Browsing by Subject "Nonsporulating strain"

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    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.