Isolation of Geobacillus stearothermophilus L-arabinose Isomerase for the Production of the Novel Food Ingredient D-tagatose

Abstract

D-tagatose, a low-calorie bulk sweetener, can be produced from D-galactose with an L-arabinose isomerase (L-AI) from Geobacillus stearothermophilus . Isolation of L-AI enzyme, intracellularly expressed in Escherichia coli , was studied by means of sonication, by chemical cell disruption and by a combined disruption approach. Conditions for cell disruption through sonication, e.g., sonication time, duty cycle, power and sample volume were determined for the wild type (WT) enzyme which was produced without an inducible expression system. The highest release was observed at a power of 120 W after 5 minutes of sonication with a duty cycle of 50% and sample volume of 60.0 mL resulting in a relative L-AI activity of 92.5 ± 0.9%. Chemical cell disruption with 16.5 mM triton X-100 for 17 hours gave even better results compared to sonication, namely, a relative L-AI activity in the crude extract of 95.6 ± 1.2%. Additional experiments were performed on cell disruption of the wild type enzyme produced with an inducible expression system (WT i ). Treatment of WT i enzyme with 16.5 mM triton X-100 for 17 hours led to a lower relative L-AI activity, namely, 84.0 ± 0.5%. A combined approach of a prior chemical lysis with triton X-100 followed by sonication led to a further increase of L-AI activity towards 89.6 ± 0.3%.