Table 3.

Relative activity of evolved CM variants

Varianta	Mutation(s)	Relative activityb
R1–2	R44C, H66R	0.5
R1–17	T61M	0.5
R1–35	T87I	1.3
R1–39	E29K	0.6
R1–78	S42L	0.8
R2–6	S15D, L79F, T87I	10
R2–7	S15N, S42L, T87I	3
R2–9	S42L, T87I	0.3
R2–12	E4K, S15D, T87I	3
R2–14	S42L, R44L, H66R	1.2
R2–26	S47L, R44L, T87I	1.4
R2–16	S15D, T87I	1.5
R2–30	E35K, S42L, T87I	0.5
R2–32	S3L, T87I	0.7

^aR1 and R2 stand for “round 1” and “round 2” variants. Kinetics were performed at 20°C in PBS (pH 7.5) using 400 nM enzyme and 100 μM chorismate. ^bThe activity of the parent hEcCM was set to 1.