Table 2.
Characterization of B. megaterium levansucrase variants.
| Products size | Variant | Position | KM (mm) | kcat (s−1) | kcat/KM (s−1 mm−1) | |||
|---|---|---|---|---|---|---|---|---|
| 370 | 373 | 419 | ||||||
| Group 1 | DP 2–20 | WTa | R | K | F | 10.8 ± 1.5 | 197.9 ± 4.0 | 18.32 |
| 3A6 | R | K | Y | — | — | — | ||
| Group 2 | DP 4 | 1H11 | H | K | F | — | — | — |
| 2B12 | H | S | F | — | — | — | ||
| 7D12b | H | K | W | — | — | — | ||
| Group 3 | DP 8 | 1G2 | R | R | F | — | — | — |
| 2H11 | R | R | Y | — | — | — | ||
| 3H5 | R | R | W | — | — | — | ||
| Group 4 | DP 4 | 2A5 | Q | K | F | 199.4 ± 15.2 | 42.7 ± 2.4 | 0.21 |
| 5F11 | Q | K | W | — | — | — | ||
| 3G5 | Q | K | Y | — | — | — | ||
| 3E7 | Q | R | W | 114.8 ± 9.1 | 24.0 ± 0.9 | 0.21 | ||
| Group 5 | DP 6–7 | 1B1 | R | H | F | 41.8 ± 6.6 | 197.5 ± 8.4 | 4.72 |
| 2E11 | R | H | Y | — | — | — | ||
| 3B9b | R | H | E | — | — | — | ||
| 3C5 | R | L | F | 12.5 ± 1.3 | 122.5 ± 2.3 | 9.80 | ||
| 4E1 | R | L | Y | — | — | — | ||
Mutated residues are shown as underlined bold letters. Catalytic parameters were only calculated for representative variants of groups 4–5. Reactions were performed in 50 mm Sörensen buffer pH 6.5, containing varying concentrations of sucrose and purified levansucrase variants. Variants not kinetically characterized are indicated by a horizontal dash (−).
aFrom16.
bThese variants display very low activity and poor sucrose conversion, producing mainly di- and trisaccharides.