2.6 Enzyme assays
WT and its mutants were assayed for their isomaltulose-forming activity
using 584 mM sucrose as substrate in 50 mM citric acid-sodium dihydrogen
phosphate buffer (pH 6.0). Specifically, the reaction mixture consisting
of 900 μL of 584 mM sucrose solution and 100 μL of purified enzymes in a
final volume of 1 mL was incubated at 30°C for 10 min. The reaction was
terminated by boiling the samples at 100℃ for 10 min. The isomaltulose
production was quantified by high-performance liquid chromatography
(HPLC). One unit (U) of enzyme activity was defined as the amount of
enzyme that catalyzed the formation of 1 μmol isomaltulose in 1 min
under the described conditions.