TABLE 1.

Determination of the E value for the enantioselective production of (R)-1-phenylethane-1,2-diol from racemic styrene oxide catalyzed by wild-type EchA and variants

Enzymea	Formation of:				Hydrolysis of racemic styrene oxideb		Enantioselectivityd
	(R)-1-phenylethane-1,2-diolb		(S)-1-phenylethane-1,2-diolc
	Rate (μmol/min/mg)	Fold increase relative to WT	Rate (μmol/min/mg)	Fold increase relative to WT	Rate (μmol/min/mg)	Fold increase relative to WT	E value	Fold increase relative to WT
Wild-type EchA	1.04 ± 0.07	1.0	1.00 ± 0.18	1.0	1.77 ± 0.18	1.0	17 ± 0.5	1.0
F108L	2.23 ± 0.01	2.1	1.41 ± 0.05	1.4	2.80 ± 0.03	1.6	9 ± 1.0	0.5
1219F	1.96 ± 0.09	1.9	0.06 ± 0.003	0.06	2.26 ± 0.28	1.3	91 ± 12	5.3
C2481	1.60 ± 0.13	1.5	0.61 ± 0.14	0.6	2.02 ± 0.04	1.1	10 ± 0	0.6
F108L/C2481	1.85 ± 0.08	1.8	0.29 ± 0.03	0.3	2.57 ± 0.23	1.4	46 ± 11	2.7
1219L/C2481	1.35 ± 0.07	1.3	0.40 ± 0.26	0.4	1.87 ± 0.13	1.0	35 ± 11	2.0
F108L/I219L/C248I	2.16 ± 0.01	2.1	0.43 ± 0.04	0.4	3.01 ± 0.36	1.7	31 ± 0.1	1.8
L190F	2.60 ± 0.32	2.5	2.46 ± 0.60	2.5	8.55 ± 0.26	4.8	2 ± 0.5	0.1
L190Y	1.94 ± 0.14	1.9	1.61 ± 0.70	1.6	4.82 ± 0.82	2.7	14 ± 0.6	0.8

^aCrude enzymes were prepared by cell sonication, and the EchA concentration from each strain was 50 μg/ml as estimated from SDS-PAGE.

^bThe formation rate of (R)-1-phenylethane-1,2-diol and the hydrolysis rate of racemic styrene oxide were determined via chiral HPLC over a linear range beginning at time zero. WT, wild type.

^cThe formation rate of (S)-1-phenylethane-1,2-diol was determined over a linear range via chiral HPLC from the time when competitive substrate inhibition ceased (different for each strain).

^dThe E values for the formation of (R)-1-phenylethane-1,2-diol were calculated using the equation E = ln{1 − c · [1 + ee(P)]}/ln{1 − c · [1 − ee(P)]} (5) at c = 0.5.