Table 2. Kinetic parameters of wild-type and site-directed mutant enzymes.
The native and mutant enzymes were purified from culture supernatants as described by Arrieta et al. [17] and quantified by the Bradford procedure using BSA as a standard. The reaction mixtures (50 μl) containing 200 mM sucrose in 0.1 M sodium acetate, pH 5.0, were incubated with 0.3 unit of purified enzyme at 30 °C. After 15 min, the reaction was stopped by heating in a boiling-water bath for 5 min. Glucose released from sucrose hydrolysis was measured by a glucose oxidase/peroxidase-coupled colorimetric reaction. The kcat was calculated using 60000 as the Mr of a functional unit of levansucrase. The Km for sucrose was determined within a substrate concentration range 1–150 mM at 30 °C and pH 5.0. The kinetic parameters were determined by non-linear regression analysis of substrate–velocity plots using the LEONORA program [46]. One unit of enzyme activity is defined as the amount of enzyme releasing 1 μmol of glucose/min based on initial-velocity measurements under the following conditions: 0.2 M sucrose in 0.1 M sodium acetate buffer, pH 5.0, at 30 °C. Values are means±S.D. (n=6).
| Strain | Mutation | kcat (min−1) | Km (mM) | kcat/Km (M−1·min−1) |
|---|---|---|---|---|
| AD5 (pALS148) | Wild-type | (3.9±0.2)×103 | 11.9±0.4 | 3.3×105 |
| AD5 (pALS192) | D135N | 1.7±0.2 | 12.3±0.9 | 1.4×102 |
| AD5 (pALS205) | C339S | 66.9±2.3 | 13.8±1.2 | 4.8×103 |
| AD5 (pALS207) | C395S | 71.8±6.4 | 13.1±1.4 | 5.5×103 |