Work

Recombinant production and characterization of a novel L-arabinose isomerase for the production of D-tagatose at acidic pH

Year: 2025

Type: article

Abstract: D-Tagatose is a promising substitute for sucrose and has a lower physiological caloric value of 12.5 kJ/g compared to sucrose (17.0 kJ/g). In this study, a novel L-arabinose isomerase (L-AI; EC 5.3.1.4) from Lentilactobacillus parakefiri DSM 10551 (L-AI-Lp) was recombinantly produced in Escherichia coli , purified and biochemically characterized for the isomerization of D-galactose to D-tagatose. The influences of temperature and pH on the activity of L-AI-Lp were compared to a protein-engineered variant of L-AI from Geobacillus stearothermophilus DSM22 (L-AI-N13), which is part of a patent held by the D-tagatose-producing company Damhert Nutrition. The production of L-AI-Lp in a bioreactor cultivation yielded 27.72 μ kat D-gal, 65 °C /L culture and the L-AI was purified 2.9-fold (specific L-AI activity of 99.6 nkat D-gal, 65 °C /mg protein ) by heat treatment and anion exchange chromatography with a yield of 71% (6557 ± 411 nkat D-gal, 65 °C ). The L-AI-Lp showed favorable characteristics compared to L-AI-N13 regarding its maximal activity at 75 °C (L-AI-N13, 60–65 °C) and relative activity of ≥70% over a broad pH range of 4–9, whereas no activity was detected for L-AI-N13 at pH ≤ 5. Finally, the applicability of L-AI-Lp and L-AI-N13 was compared by the isomerization of D-galactose (300 mmol/L) at pH 7.5, whereby 45% and 41%, respectively, were isomerized after 24 h. Only the L-AI-Lp was able to isomerize 45% of D-galactose at pH 4.5 after 24 h. In conclusion, the L-AI-Lp is a promising new L-AI for industrial production of D-tagatose over a broad pH range, especially at acidic pH. • New acidophilic L-AI from Lentilactobacillus parakefiri DSM 10551 (L-AI-Lp). • L-AI-Lp is active at a broad pH range (relative activity ≥70% at pH 4.0–9.0). • L-AI-Lp showed advantageous characteristics compared to protein-engineered L-AI-N13.

Source: Food Bioscience

DOI: 10.1016/j.fbio.2025.106147

Authors: Nathanael Weber, Sabine Lutz-Wahl, Lutz Fischer

Institution: University of Hohenheim

Language: English

Topic: Enzyme Production and Characterization

Subfield: Biotechnology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $3,380

Funders: Bundesministerium für Wirtschaft und Klimaschutz, Universität Hohenheim