Table 1.
Comparison of the reaction activities, AA-2G yields, and stability of the wild-type and seven mutant CGTasesa
| CGTase | Relative activity (%)b |
AA-2G yield (g/liter)c |
t1/2 (h) at: |
|||
|---|---|---|---|---|---|---|
| Cyclization (α-cyclodextrin-forming activity) | Hydrolysis (starch-degrading activity) | Disproportionation | 40°C | pH 5.5 | ||
| Wild type | 100 | 100 | 100 | 1.01 ± 0.05 | 7.6 ± 0.4 | 29.8 ± 0.9 |
| Y260R | ND | 226 ± 10 | 138.0 ± 1.0 | 1.44 ± 0.03 | 8.0 ± 0.8 | 29.3 ± 0.7 |
| Q265K | 15 ± 0.5 | 236 ± 6 | 130.7 ± 0.8 | 1.40 ± 0.07 | 7.8 ± 0.7 | 33.6 ± 1.1 |
| Y195S | ND | 200 ± 8 | 102.6 ± 0.5 | 1.23 ± 0.04 | 8.8 ± 0.3 | 31.3 ± 0.8 |
| Y260R/Q265K | 8 ± 0.7 | 213 ± 8 | 129.2 ± 0.8 | 1.55 ± 0.05 | 8.5 ± 0.6 | 30.2 ± 0.9 |
| Y260R/195S | ND | 492 ± 7 | 142.3 ± 0.8 | 1.57 ± 0.07 | 7.9 ± 0.5 | 28.9 ± 0.6 |
| Q265K/Y195S | ND | 498 ± 9 | 140.1 ± 0.7 | 1.49 ± 0.08 | 7.7 ± 0.7 | 30.4 ± 1.0 |
| Y260R/Q265K/195S | 12 ± 0.9 | 557 ± 5 | 148.0 ± 0.6 | 1.59 ± 0.05 | 7.9 ± 0.4 | 31.7 ± 0.5 |
a
Each value represents the mean of triple independent measurements, and the deviation from the mean was below 5%. t1/2, half-life.
b
The cyclization, hydrolysis, and disproportionation reaction activities for wild-type CGTase was 165 ± 5, 8.3 ± 0.1, and 806 ± 8 U/mg, respectively, and were defined as 100% for the relative activity. ND, not detectable.
c
Maltodextrin was the glycosyl donor.