TABLE 1.
Characterization of the engineered enzymesa
| Acylase | Substituted site(s) | Km (mM) | kcat (s−1) | Catalysis efficiency (kcat/Km [M−1/s−1]) | Total activity (U/liter) | Sp act (U/mg) |
|---|---|---|---|---|---|---|
| CA130 | Native acylase | 0.45 | 12.3 | 2.73 × 104 | 850 | 11.8 |
| Y151αF | Tyr151α→Phe | 0.24 | 14.4 | 6.00 × 104 | 1,050 | 14.4 |
| Q50βN | Gln50β→Asn | 0.22 | 17.9 | 8.14 × 104 | 1,250 | 17.5 |
| Y151αF/Q50βN | Tyr151α→Phe and Gln50β→Asn | 0.38 | 13.4 | 3.53 × 104 | 900 | 12.3 |
| R121βA | Arg121β→Ala | 0.45 | 12.2 | 2.71 × 104 | 840 | 11.6 |
| K198βA | Lys198β→Ala | 0.47 | 11.7 | 2.49 × 104 | 815 | 11.3 |
| D286βA | Asp286β→Ala | 0.41 | 12.7 | 3.10 × 104 | 870 | 12.0 |
| Q50βN/K198βA | Gln50β→Asn and Lys198β→Ala | 0.21 | 17.8 | 8.48 × 104 | 1,235 | 17.4 |
a
All of the enzymes for which activities were assayed for calculating the kinetic parameters were purified as described in Materials and Methods. Data are mean values of five determinations from independent experiments.