Characterisation of immobilised Geobacillus stearothermophilus L-arabinose isomerase

The L-arabinose isomerase from Geobacillus stearothermophilus was studied for the production of D-tagatose, a low-calorie bulk sweetener. Industrially, D-tagatose is only produced in a chemical way. However, a lot of waste and by-products are formed in this way. A biological synthesis of D-tagatose is not yet commercially available. In this work first, L-arabinose isomerase was intracellularly expressed in E. coli. The enzyme was isolated and purified. Hereby, three different L-arabinose isomerase fractions were obtained, namely the intracellular enzyme, crude extract and purified enzyme. This work is first to characterise and compare three different immobilised enzyme fractions from wild type Geobacillus stearothermophilus in a systematic approach. The L-AI activity was determined of the different enzyme fractions obtained during the isolation and purification. The L-AI activity in the intracellular enzyme, crude extract and purified enzyme were 8.55 ± 0.13 U/ml, 7.95 ± 0.03 U/ml and 6.75 ± 0.06 U/ml, respectively. Furthermore, prior to immobilisation, the intracellular enzyme and crude extract were diluted to the same protein concentration as the purified enzyme. The diluted intracellular enzyme, diluted crude extract and purified enzyme were prior to the immobilisation experiment, subjected to a conversion experiment at 60°C and pH 7.0. After 48 hours of incubation, D-tagatose concentrations of 67.2 ± 0.8 g/l, 61.3 ± 3.2 g/l and 68.7 ± 2.2 g/l were found, respectively. Conversion to D-tagatose was proven for all the enzyme fractions. Next, the three enzyme fractions were entrapped in alginate beads and the optimal temperature and pH for the conversion of D-galactose to D-tagatose were determined. Experiments were carried out with temperatures ranging from 50 to 90°C at a pH of 7.0. For the optimal pH, experiments were carried out from pH 5.0 to pH 9.0. The D-tagatose concentrations were determined using cysteine-carbazole method. The different immobilised enzyme fractions showed an optimal temperature of 80°C. After 5 hours of incubation at 80°C and pH 7.0, a D-tagatose concentration of 54.2 ± 0.5 g/l, 27.9 ± 1.2 g/l and 36.1 ± 1.0 g/l was obtained for the diluted intracellular enzyme, diluted crude extract and purified enzyme, respectively. The optimal pH was 6.5 for the different beads. After 5 hours of incubation at 60°C and pH 6.5, a D-tagatose concentration of 23.2 ± 0.3 g/l, 10.5 ± 0.3 g/l and 13.2 ± 0.3 g/l was obtained for diluted intracellular enzyme, diluted crude extract and purified enzyme, respectively. The results within this study revealed that intracellular enzyme, crude extract and purified L-arabinose isomerase entrapped in alginate beads have the same optimal operational conditions for the conversion of D-galactose into D-tagatose. An optimal temperature and pH of 80°C and 6.5 were determined for each fraction individually. These findings will be useful for the biological conversion of D-galactose to D-tagatose. This is a first step of a bigger research to improve the productivity and stability of immobilised L-AI from Geobacillus stearothermophilus for the production of D-tagatose.