TABLE 1.
Specific activities of wild-type and mutant lipasesa
| Lipase | Activity (U/ml) | Protein concn (10−3 mg/ml) | Sp act (103 U/mg) |
|---|---|---|---|
| WT | 101.4 ± 3.5 | 6.9 ± 0.35 | 14.7 ± 1.3 |
| C190S | 2.20 ± 0.14 | 4.0 ± 0.22 | 0.55 ± 0.07 |
| C270S | 3.66 ± 0.11 | 3.8 ± 0.13 | 0.96 ± 0.07 |
| C190/270S | 3.05 ± 0.26 | 2.4 ± 0.05 | 1.27 ± 0.14 |
| D242A | 5.49 ± 0.08 | 2.4 ± 0.02 | 2.29 ± 0.05 |
| D288A | 7.08 ± 0.32 | 6.4 ± 0.29 | 1.11 ± 0.10 |
| D242/288A | 3.17 ± 0.13 | 2.8 ± 0.06 | 1.13 ± 0.07 |
a
Wild-type (WT) and mutant lipases were expressed by the coupled transcription-translation system with the PCR-amplified DNA as templates in the presence of presynthesized activator. Lipase activities and protein concentrations of the samples from 60 min of translation reaction were analyzed. Protein concentration was deduced from the radioactivity of [14C]leucine incorporated into the synthesized lipase.