D tagatose

Example 11

The recombinant protein having isomerase activity prepared from E. coli was immobilized by using techniques known in the prior art or directly contacted with D-galactose solution for production of D-tagatose. The bioconversion conditions comprise maintaining the D-galactose substrate concentration between 20% and 95% (w/v) and 100 to 1000 units of the recombinant isomerase. For the present embodiment, the substrate was taken at a concentration of 100 g/L and 100 Units of immobilized enzyme was used. Bioconversion reaction was carried out in 20 mM Tris-HCl buffer containing 5 mM MnCl₂ at pH 8.0 at temperature between 60° C. The conversion of D-allulose to D-allose reached saturation at higher substrate concentration, preferably between 40% to 50% (w/v) at enzyme concentration between 400 to 600, preferably 500 units of enzyme with reaction time of about 6 h.

The reaction mixture was subjected to HPLC analysis to confirm the residual substrate and product formation. The product peak was confirmed with commercially available D-galactose and D-tagatose (Sigma Aldrich) as substrate and product standards, respectively. The results of the studies are depicted in FIGS. 9A-9C. FIG. 9A depicts D-galactose standard chromatogram, FIG. 9B depicts D-tagatose standard chromatogram and FIG. 9C depicts D-galactose and D-tagatose bioconversion mixture chromatogram.