Overexpression of L-Arabinose Isomerase for Production of the Low-Calorie Bulk Sweetener D-Tagatose

Abstract

High-Cell-Density Cultivations show a huge potential to produce recombinant proteins to amounts greatly exceeding the availability in natural resources. An interesting example of a recombinant protein is an L-arabinose isomerase, which is able to convert D-galactose to the low-caloric and low-glycaemic bulk sweetener D-tagatose. Within this study, the L-arabinose isomerase from Geobacillus stearothermophilus was expressed intracellularly in Escherichia coli. The cultivation medium contained glucose, yeast extract and various macro- and micronutrients. The effect of air flow rate on E. coli growth and expression of L-arabinose isomerase was studied. After 52 hours, an Optical Density and Dry Cell Weight of 154 ± 4 and 54.8 ± 1.3 g L-1 were reached, respectively by regulating the air flow rate between 0.2 and 30 L min-1. A corresponding L-arabinose isomerase activity of 6.99 ± 0.46 U mL-1 was reached. A drawback of High-Cell-Density Cultivation is the production of the by-product acetic acid which may inhibit growth. However, the acetic acid concentration was maintained as low as possible during fermentation to avoid inhibitory effects inherent to this compound. With the L-arabinose isomerase produced, a conversion percentage of 37.1 ± 1.5% was achieved, corresponding to 94.9 ± 3.7 g L-1 D-tagatose. Thus, the implementation of a High-CellDensity Cultivation led to an efficient expression of the L-arabinose isomerase enzyme and D-tagatose production. Also the storage stability of the cells was investigated during several months at 4°C. A stable L-arabinose isomerase enzyme was noticed during at least 8 months storage at 4°C.