Substrate specificity of a galactose 6-phosphate isomerase from Lactococcus lactis that produces d-allose from d-psicose

We purified recombinant galactose 6-phosphate isomerase (LacAB) from Lactococcus lactis using HiTrap Q HP and Phenyl-Sepharose columns. The purified LacAB had a final specific activity of 1.79 units/mg to produce d-allose. The molecular mass of native galactose 6-phosphate isomerase was estimated at 135.5 kDa using Sephacryl S-300 gel filtration, and the enzyme exists as a hetero-octamer of LacA and LacB subunits. The activity of galactose 6-phosphate isomerase was maximal at pH 7.0 and 30 °C, and enzyme activity was independent of metal ions. When 100 g/L of d-psicose was used as the substrate, 25 g/L of d-allose and 13 g/L of d-altrose were simultaneously produced at pH 7.0 and 30 °C after 12 h of incubation. The enzyme had broad specificity for various aldoses and ketoses. The interconversion of sugars with the same configuration except at the C2 position was driven by using a large amount of enzyme in extended reactions. The interconversion occurred via two isomerization reactions, i.e., the interconversion of d-allose ↔ d-psicose ↔ d-altrose, and d-allose to d-psicose reaction was faster than d-altrose to d-psicose reaction.