TABLE 2.
Characterization of the wild-type HheC and the enantioselective mutants obtained from combinatorial libraries
| Enzyme | Absolute configuration |
rac-2-Chloro-1-phenylethanol |
(R)-2-Chloro-1-phenylethanol |
(S)-2-Chloro-1-phenylethanol |
|||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| eep (%) | % conversion (min) | E valuea | Km (mM) | kcat (s−1) | kcat/Km (s−1 M−1) | Km (mM) | kcat (s−1) | kcat/Km (s−1 M−1) | Ecalb | ||
| HheC WT | R | 91 | 51 (20) | 65 | 0.45 ± 0.04 | 14.83 ± 1.40 | 32,956 | 4.53 ± 0.46 | 2.37 ± 0.23 | 523 | 63 |
| P84V/F86P mutant | R | 85 | 48 (20) | 23 | 0.72 ± 0.13 | 25.1 ± 1.89 | 34,861 | 2.19 ± 0.61 | 4.24 ± 1.11 | 1,936 | 18 |
| P84V/F86P/N176A mutant | S | 84 | 50 (7.5) | 28 | 2.54 ± 0.43 | 6.57 ± 0.85 | 2,587 | 0.60 ± 0.09 | 29.98 ± 2.1 | 49,967 | 19 |
| P84V/F86P/T134C/N176A mutant | S | 90 | 51 (25) | 54 | 6.73 ± 0.58 | 3.54 ± 0.32 | 526 | 0.52 ± 0.11 | 13.56 ± 1.21 | 26,077 | 50 |
| P84V/F86P/T134A/N176A mutant | S | 97 | 49 (20) | 101 | 8.02 ± 1.03 | 2.77 ± 0.20 | 345 | 0.42 ± 0.06 | 15.06 ± 1.03 | 35,857 | 104 |
| T134V/L142M mutant | R | 97 | 47 (40) | 107 | 0.23 ± 0.07 | 5.61 ± 0.63 | 24,391 | 9.70 ± 1.18 | 2.19 ± 0.17 | 226 | 108 |
| L142F/N176H mutant | R | 98 | 57 (50) | 132 | 0.19 ± 0.05 | 4.45 ± 0.37 | 23,421 | 10.31 ± 1.01 | 1.57 ± 0.09 | 162 | 152 |
| P84V/F86P/T134A/W139Q/N176A/Y187F mutant | S | 94 | 36 (40) | 54 | —c | — | — | — | — | — | — |
a
E values were calculated by using the program Selectivity (K. Faber and H. Hoenig [ftp://borgc185.kfunigraz.ac.at/pub/enantio/]).
b
The Ecal value was calculated using the equation Ecal = (kcat/Km)R/(kcat/Km)S (R preference mutants) or Ecal = (kcat/Km)S/(kcat/Km)R (S preference mutants).
c
—, not determined.