Table 2.
Kinetic parameters for the Bacillus circulans ATCC 31382 β-galactosidase rGalD-D and the E532Q site-directed mutant.a All parameters were determined at 40 °C and pH 6.0. One enzyme activity unit is defined as the amount of enzyme in mg required for the hydrolysis of 1 μmol lactose per min at 40 °C and pH 6.0. Hydrolysis of oNPG (oNP release at 420 nm) was used to determine the kinetic parameters Vmax (μmol min−1 mg−1 of protein), KM (mM) and kcat (s−1). Subscripts high and low indicate parameters determined from the high and low substrate regions in biphasic Lineweaver–Burk plots, respectively. All measurements were done in triplo.
| Enzyme | rGalD-D | E532Q |
|---|---|---|
| Lactose hydrolysis units min−1 mg−1 | 158.5 ± 5.4 | 0.47 ± 0.02 |
| oNPG hydrolysis kinetic parameters | ||
| Vmax,high | 277.7 ± 2.7 | 1.0 ± 0.1 |
| Km,high | 45.9 ± 2.7 | 43.6 ± 0.1 |
| kcat,high | 424.1 ± 4.1 | 1.6 ± 0.1 |
| Vmax,low | 35.8 ± 9.3 | 0.2 ± 0.01 |
| Km,low | 1.5 ± 0.9 | 1.9 ± 0.3 |
| kcat,low | 56.3 ± 11.9 | 0.2 ± 0.01 |
a
H379F, H345F and E447N site-directed mutants were inactive.