D2D CURE | Data | Protein Characterization Data

BglB Variant Characterization Data

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☑ Rosetta/Foldit sequence numbering.
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1288 records found

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.pbb structural image

Color Key
relative scale	−5		−4		−3		−2		−1		0		1

For kinetic constants,
the table is color-coded by relative log values of
1/K_M, k_cat, and k_cat/K_M compared to WT.
log 1/K_M is used so that larger values are "better".

For T₅₀ and T_M values and Rosetta scores, a linear scale is used.

Blue shading indicates a variant that is "worse" than WT.
Orange shading indicates a variant that is "better" than WT.

Variants shaded black expressed (as confirmed by gel electrophoresis and/or yield > 0.1 mg/mL).
Variants marked with an asterisk (*) expressed, but no yield was recorded.

How were these data calculated?

Variant ▲	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change	ref(s)
WT	*	5.00 ± 0.20	720.9 ± 8.7	144.18 ± 6.06	39.9 ± 0.1	45.2 ± 1.3	—	¹, ², ³
WT	0.21	5.70 ± 0.38	707.7 ± 12.6	124.14 ± 8.59	—	—	—	³
WT	1.03	4.42 ± 0.84	666.7 ± 32.0	150.72 ± 29.38	—	—	—	³
WT	1.16	6.63 ± 1.23	384.2 ± 19.6	57.98 ± 11.14	—	—	—	³
WT	0.88	6.22 ± 0.48	625.3 ± 13.1	100.56 ± 8.05	—	—	—	³
WT	0.10	4.50 ± 0.76	352.5 ± 15.2	78.27 ± 13.67	—	—	—
WT	0.28	2.91 ± 0.83	272.7 ± 17.2	93.81 ± 27.29	—	—	—
WT	1.46	3.25 ± 0.59	617.5 ± 26.9	189.84 ± 35.57	—	—	—
WT	1.38	1.22 ± 0.19	317.5 ± 10.7	261.23 ± 42.37	40.8 ± 0.3	—	—
WT	0.83	3.86 ± 1.47	426.0 ± 41.2	110.49 ± 43.36	40.3 ± 0.2	—	—
WT	0.83	6.47 ± 0.41	622.5 ± 11.0	96.22 ± 6.29	40.3 ± 0.2	—	—
WT	0.83	4.04 ± 0.49	471.9 ± 14.6	116.70 ± 14.55	40.1 ± 0.1	—	—
WT	0.83	5.17 ± 0.27	571.6 ± 8.0	110.52 ± 5.98	40.6 ± 0.1	—	—
WT	0.83	2.98 ± 0.44	405.4 ± 18.2	135.87 ± 21.06	—	—	—
WT	0.83	0.63 ± 0.26	375.4 ± 28.8	600.61 ± 253.61	—	—	—
WT	0.44	4.17 ± 0.09	272.0 ± 1.5	65.17 ± 1.46	—	—	—
WT	3.71	4.09 ± 0.42	361.1 ± 9.5	88.21 ± 9.34	—	—	—
WT	4.36	11.33 ± 0.98	304.6 ± 8.1	26.88 ± 2.44	37.7 ± 0.4	—	—
WT	3.17	10.97 ± 0.99	339.8 ± 9.8	30.98 ± 2.94	38.5 ± 0.3	—	—
WT	8.69	8.25 ± 0.76	163.0 ± 4.5	19.76 ± 1.90	36.2 ± 0.2	—	—
WT	4.69	16.80 ± 1.40	256.9 ± 7.5	15.29 ± 1.35	36.8 ± 0.3	—	—
WT	1.20	8.56 ± 0.73	135.5 ± 3.5	15.82 ± 1.41	36.8 ± 0.2	—	—
WT	1.54	11.72 ± 0.78	204.0 ± 4.4	17.40 ± 1.22	37.4 ± 0.1	—	—
WT	2.17	—	—	—	38.9 ± 0.2	—	—
WT	1.55	4.74 ± 0.48	698.0 ± 18.6	147.11 ± 15.31	38.9 ± 0.3	—	—
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
WT	2.24	1.59 ± 0.16	507.3 ± 11.3	318.68 ± 33.03	37.0 ± 0.5	—	—
WT	0.25	2.47 ± 0.47	333.9 ± 14.9	135.04 ± 26.20	38.3 ± 0.3	—	—
WT	2.17	4.19 ± 0.74	720.0 ± 31.0	171.63 ± 31.15	39.1 ± 0.2	—	—
WT	2.96	3.22 ± 0.20	636.5 ± 9.7	197.96 ± 12.59	39.3 ± 0.2	—	—
WT	2.17	4.19 ± 0.74	720.0 ± 31.0	171.63 ± 31.15	39.1 ± 0.2	—	—
WT	1.73	6.12 ± 0.27	348.5 ± 4.3	56.93 ± 2.60	—	—	—
WT	4.18	2.98 ± 0.38	240.5 ± 7.5	80.76 ± 10.74	—	—	—
WT	2.96	3.22 ± 0.20	636.5 ± 9.7	197.96 ± 12.59	—	—	—
WT	1.55	4.74 ± 0.48	698.0 ± 18.6	147.11 ± 15.31	—	—	—
WT	2.23	2.90 ± 0.31	480.8 ± 12.4	165.73 ± 18.21	—	—	—
WT	3.45	1.51 ± 0.22	389.5 ± 11.8	257.63 ± 38.03	—	—	—
WT	3.59	6.37 ± 0.51	411.6 ± 9.2	64.57 ± 5.35	—	—	—
WT	4.17	3.01 ± 0.31	231.6 ± 5.7	76.97 ± 8.09	—	44.7 ± 0.3	—
WT	2.30	5.00 ± 0.90	324.7 ± 14.9	64.92 ± 12.10	—	—	—
WT	5.60	4.69 ± 0.61	210.4 ± 8.2	44.83 ± 6.12	39.0 ± 0.3	—	—
WT	3.33	3.78 ± 0.34	339.4 ± 7.7	89.80 ± 8.33	—	—	—
WT	2.03	2.02 ± 0.28	150.5 ± 4.7	74.34 ± 10.46	39.7 ± 0.2	—	—
WT	1.36	1.86 ± 0.46	162.4 ± 9.7	87.35 ± 22.41	40.1 ± 0.4	—	—
WT	5.45	3.75 ± 0.40	180.8 ± 4.9	48.24 ± 5.34	38.6 ± 0.3	—	—
WT	1.76	3.70 ± 0.58	356.2 ± 14.2	96.38 ± 15.66	—	—	—
WT	4.55	4.36 ± 0.18	688.0 ± 7.3	157.97 ± 6.69	—	—	—
WT	3.31	2.41 ± 0.52	246.5 ± 11.9	102.37 ± 22.85	—	—	—
WT	3.56	3.93 ± 0.26	860.6 ± 14.5	218.86 ± 14.93	—	—	—
WT	3.25	4.92 ± 0.50	284.9 ± 7.7	57.89 ± 6.07	39.2 ± 0.2	—	—
WT	3.25	4.92 ± 0.50	284.9 ± 7.7	57.89 ± 6.07	39.1 ± 0.3	—	—
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
WT	1.07	6.09 ± 0.51	264.4 ± 6.2	43.43 ± 3.80	40.5 ± 0.3	—	—
WT	1.07	6.09 ± 0.51	264.4 ± 6.2	43.43 ± 3.80	—	—	—
WT	2.27	11.32 ± 2.41	291.4 ± 18.8	25.73 ± 5.71	40.8 ± 0.1	—	—
WT	3.81	3.38 ± 0.30	231.1 ± 5.1	68.28 ± 6.25	—	—	—
WT	3.75	7.03 ± 0.52	264.2 ± 5.6	37.56 ± 2.90	—	—	—
WT	3.75	6.76 ± 0.31	453.7 ± 5.9	67.16 ± 3.20	—	—	—
WT	0.58	6.74 ± 0.61	229.2 ± 5.9	34.00 ± 3.22	—	—	—
WT	0.58	6.74 ± 0.61	229.2 ± 5.9	34.00 ± 3.22	—	45.5 ± 0.3	—
WT	2.59	5.99 ± 0.53	275.7 ± 6.8	46.00 ± 4.25	—	46.6 ± 0.1	—
WT	2.55	5.00 ± 0.45	367.4 ± 8.7	73.47 ± 6.78	—	—	—
WT	2.72	4.36 ± 0.34	342.2 ± 6.7	78.56 ± 6.28	—	46.3 ± 0.2	—
WT	0.58	6.74 ± 0.61	229.2 ± 5.9	34.00 ± 3.22	—	45.5 ± 0.3	—
WT	2.83	4.43 ± 0.38	372.3 ± 8.3	84.04 ± 7.38	—	47.6 ± 1.5	—
WT	0.71	4.22 ± 0.54	295.6 ± 9.8	70.03 ± 9.28	—	47.0 ± 0.3	—
WT	2.87	4.38 ± 0.39	368.6 ± 8.6	84.18 ± 7.79	—	45.4 ± 0.1	—
WT	2.84	3.99 ± 0.40	288.8 ± 7.4	72.44 ± 7.55	—	44.4 ± 0.2	—
WT	0.83	3.69 ± 0.41	401.0 ± 11.3	108.59 ± 12.56	41.1 ± 0.1	—	—
WT	3.26	5.92 ± 0.21	424.4 ± 4.2	71.73 ± 2.64	—	—	—
WT	2.60	3.07 ± 0.40	388.2 ± 12.5	126.65 ± 17.19	40.3 ± 0.2	—	—
WT	3.59	6.54 ± 0.86	353.5 ± 14.0	54.08 ± 7.41	—	47.1 ± 0.2	—
WT	0.87	3.88 ± 0.21	261.9 ± 3.5	67.45 ± 3.68	42.2 ± 0.7	—	—
WT	0.30	6.77 ± 0.79	216.5 ± 7.1	31.97 ± 3.89	—	45.9 ± 0.3	—
WT	4.24	7.23 ± 0.71	488.6 ± 13.9	67.61 ± 6.95	—	—	—
WT	2.80	4.89 ± 0.41	272.4 ± 6.1	55.74 ± 4.85	—	44.4 ± 0.1	—
WT	1.17	3.19 ± 0.23	403.2 ± 7.3	126.48 ± 9.53	—	46.0 ± 0.5	—
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
WT	2.91	4.29 ± 0.28	226.1 ± 3.9	52.77 ± 3.62	—	45.2 ± 0.5	—
WT	2.48	4.07 ± 0.25	326.4 ± 5.1	80.16 ± 5.02	—	—	—
WT	0.31	8.86 ± 1.29	106.8 ± 4.7	12.05 ± 1.83	39.0 ± 1.1	—	—
WT	0.31	6.48 ± 0.99	867.7 ± 37.0	133.97 ± 21.33	37.3 ± 0.3	—	—
WT	0.98	4.08 ± 0.32	244.0 ± 4.9	59.85 ± 4.88	41.5 ± 2.5	—	—
WT	1.79	5.98 ± 0.47	398.5 ± 8.2	66.66 ± 5.41	39.9 ± 1.1	—	—
WT	*	—	—	—	39.5 ± 0.2	—	—
WT	0.60	2.80 ± 0.16	362.9 ± 4.9	129.46 ± 7.44	—	—	—
WT	9.88	2.20 ± 0.26	123.8 ± 3.4	56.31 ± 6.96	39.4 ± 0.3	—	—
WT	2.29	5.46 ± 0.42	169.2 ± 3.6	30.99 ± 2.49	39.3 ± 0.3	—	—
WT	0.31	8.86 ± 1.29	1,067.7 ± 46.9	120.54 ± 18.33	39.0 ± 1.1	—	—
WT	0.31	6.48 ± 0.99	867.7 ± 37.0	133.97 ± 21.33	37.3 ± 0.3	—	—
WT	0.31	7.94 ± 1.14	1,050.9 ± 43.9	132.37 ± 19.74	38.5 ± 0.7	—	—
WT	6.51	5.51 ± 0.24	173.5 ± 2.0	31.50 ± 1.40	38.9 ± 0.1	—	—
WT	6.88	5.15 ± 0.21	182.2 ± 2.0	35.38 ± 1.51	39.6 ± 1.4	—	—
WT	0.56	4.56 ± 0.59	349.6 ± 11.8	76.67 ± 10.19	39.5 ± 0.9	—	—
WT	1.24	2.49 ± 0.22	327.1 ± 7.0	131.32 ± 12.13	39.7 ± 0.5	—	—
WT	1.31	4.50 ± 1.10	389.2 ± 25.0	86.50 ± 21.92	39.1 ± 0.3	—	—
WT	0.09	4.12 ± 0.38	162.3 ± 3.8	39.43 ± 3.71	39.8 ± 0.3	—	—
WT	0.26	5.09 ± 0.43	452.0 ± 10.2	88.86 ± 7.79	—	—	—
WT	0.31	3.82 ± 0.06	360.7 ± 1.9	94.53 ± 1.66	—	—	—
WT	1.84	9.35 ± 2.27	432.1 ± 35.2	46.22 ± 11.82	—	—	—
WT	2.53	4.15 ± 0.33	347.5 ± 7.1	83.70 ± 6.86	—	45.1 ± 0.3	—
WT	1.14	7.41 ± 0.88	1,527.6 ± 52.4	206.09 ± 25.39	38.3 ± 0.5	—	—
WT	1.14	7.76 ± 0.92	1,553.0 ± 53.9	200.21 ± 24.75	37.9 ± 0.6	—	—
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
WT	1.14	45.30 ± 6.40	1,586.7 ± 108.8	35.03 ± 5.50	38.5 ± 0.3	—	—
WT	1.14	29.90 ± 3.79	1,353.3 ± 72.7	45.25 ± 6.23	38.6 ± 0.5	—	—
WT	1.14	24.20 ± 4.99	1,340.1 ± 105.1	55.38 ± 12.21	37.5 ± 1.6	—	—
WT	1.01	4.17 ± 0.37	177.3 ± 4.0	42.55 ± 3.86	41.0 ± 0.3	—	—
WT	1.68	2.89 ± 0.15	107.4 ± 1.4	37.18 ± 2.05	39.2 ± 0.3	—	—
WT	1.68	3.64 ± 0.24	152.5 ± 2.5	41.87 ± 2.80	39.4 ± 0.2	—	—
WT	1.31	4.13 ± 0.27	157.0 ± 2.6	38.03 ± 2.55	39.3 ± 1.1	—	—
WT	1.16	4.04 ± 0.86	207.2 ± 12.0	51.30 ± 11.35	39.4 ± 0.5	—	—
WT	0.78	5.74 ± 0.45	401.2 ± 8.6	69.94 ± 5.65	—	43.4 ± 0.1	—
WT	0.72	3.76 ± 0.16	175.0 ± 2.0	46.56 ± 2.03	41.0 ± 0.2	—	—
WT	1.19	4.12 ± 0.26	666.2 ± 10.9	161.71 ± 10.58	—	—	—
WT	1.19	4.28 ± 0.21	678.0 ± 8.5	158.24 ± 7.86	—	—	—
WT	*	—	—	—	—	—	—
WT	1.60	8.50 ± 1.59	564.2 ± 20.8	66.40 ± 12.65	40.4 ± 0.2	—	—
WT	0.50	4.52 ± 0.42	1,664.1 ± 40.3	368.43 ± 35.24	41.5 ± 5.2	—	—
WT	0.50	2.20 ± 0.31	294.6 ± 9.7	133.76 ± 19.59	—	—	—
WT	0.65	2.62 ± 0.40	246.7 ± 9.5	94.05 ± 14.90	41.3 ± 0.4	45.7 ± 0.0	—
WT	0.65	3.93 ± 0.36	274.1 ± 6.5	69.74 ± 6.65	40.3 ± 0.4	45.7 ± 0.0	—
WT	0.65	2.62 ± 0.40	246.7 ± 9.5	94.05 ± 14.90	40.3 ± 0.4	45.7 ± 0.0	—
WT	1.64	4.70 ± 0.28	697.5 ± 10.9	148.30 ± 9.04	39.8 ± 0.2	—	—
WT	1.64	5.40 ± 0.32	752.9 ± 12.0	139.43 ± 8.49	39.5 ± 0.4	—	—
WT	1.50	10.14 ± 4.39	1,138.8 ± 153.9	112.35 ± 51.00	—	—	—
WT	3.88	8.83 ± 0.27	377.8 ± 3.5	42.79 ± 1.37	—	47.5 ± 0.2	—
WT	5.18	6.12 ± 0.38	366.7 ± 6.4	59.88 ± 3.90	—	45.4 ± 0.0	—
WT	4.66	6.06 ± 0.69	467.4 ± 14.6	77.19 ± 9.10	—	46.1 ± 0.0	—
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
WT	2.89	3.03 ± 0.34	275.7 ± 7.5	91.12 ± 10.48	—	43.8 ± 0.0	—
WT	4.55	7.03 ± 0.31	382.0 ± 4.9	54.35 ± 2.53	—	42.4 ± 0.0	—
WT	1.36	5.81 ± 0.38	1,017.5 ± 18.0	175.22 ± 11.89	—	43.5 ± 0.2	—
WT	*	—	—	—	—	42.4 ± 0.0	—
WT	2.11	4.31 ± 0.57	451.1 ± 15.2	104.60 ± 14.34	—	44.9 ± 0.3	—
WT	0.99	8.42 ± 1.40	489.1 ± 24.2	58.08 ± 10.06	—	43.5 ± 0.1	—
WT	0.60	8.76 ± 0.45	302.9 ± 4.7	34.60 ± 1.85	38.4 ± 0.2	—	—
WT	1.02	5.38 ± 0.37	194.1 ± 3.6	36.04 ± 2.58	39.3 ± 0.2	—	—
WT	4.54	4.99 ± 0.43	207.1 ± 4.7	41.53 ± 3.67	—	—	—
WT	1.68	5.61 ± 0.43	162.9 ± 3.4	29.03 ± 2.30	38.1 ± 0.2	—	—
WT	5.47	3.52 ± 0.34	151.7 ± 3.6	43.04 ± 4.26	39.5 ± 0.3	—	—
WT	2.18	4.23 ± 0.26	137.4 ± 2.2	32.47 ± 2.07	38.9 ± 0.3	—	—
WT	5.04	3.30 ± 0.33	149.3 ± 3.7	45.31 ± 4.72	39.5 ± 0.4	—	—
WT	0.31	6.63 ± 0.42	723.2 ± 12.4	109.01 ± 7.16	—	—	—
WT	2.03	2.91 ± 0.34	112.1 ± 3.2	38.45 ± 4.62	38.3 ± 0.5	—	—
WT	1.51	6.36 ± 1.66	100.4 ± 7.5	15.78 ± 4.29	38.8 ± 0.2	—	—
WT	0.75	—	—	—	39.8 ± 0.4	—	—
WT	2.82	5.24 ± 0.51	110.3 ± 2.8	21.08 ± 2.14	40.2 ± 0.4	—	—
WT	6.24	4.78 ± 0.16	184.6 ± 1.6	38.60 ± 1.32	39.7 ± 0.5	—	—
WT	3.68	7.35 ± 0.49	302.3 ± 5.8	41.13 ± 2.84	—	44.6 ± 0.2	—
WT	1.70	4.40 ± 0.32	402.7 ± 7.7	91.59 ± 6.98	40.2 ± 0.2	—	—
WT	1.40	3.59 ± 0.77	762.0 ± 41.1	212.51 ± 47.08	41.2 ± 0.4	—	—
WT	2.50	3.60 ± 0.15	440.5 ± 4.7	122.54 ± 5.37	40.2 ± 0.5	—	—
WT	1.40	3.92 ± 0.47	377.2 ± 11.4	96.32 ± 11.83	42.1 ± 0.4	—	—
WT	0.90	2.07 ± 0.29	251.8 ± 8.2	121.44 ± 17.72	39.4 ± 0.3	—	—
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
WT	1.70	1.63 ± 0.61	190.3 ± 16.4	116.93 ± 45.24	39.8 ± 0.4	—	—
WT	1.70	2.36 ± 0.92	252.5 ± 23.1	106.78 ± 42.65	40.1 ± 0.5	—	—
WT	1.50	2.32 ± 0.29	685.8 ± 20.3	295.82 ± 38.59	38.2 ± 0.4	—	—
WT	4.09	6.62 ± 0.49	400.3 ± 8.5	60.45 ± 4.70	—	43.2 ± 0.0	—
WT	5.04	3.30 ± 0.33	149.3 ± 3.7	45.31 ± 4.72	39.4 ± 0.4	39.4 ± 0.4	—
WT	2.10	5.64 ± 0.45	430.7 ± 9.5	76.31 ± 6.35	—	—	—
WT	0.86	4.29 ± 0.80	723.3 ± 34.8	168.79 ± 32.36	41.0 ± 0.4	—	—
WT	1.20	5.98 ± 1.75	1,423.4 ± 108.7	238.05 ± 71.98	40.9 ± 2.9	—	—
WT	1.44	4.66 ± 0.68	707.1 ± 27.3	151.73 ± 22.98	40.7 ± 0.2	—	—
WT	1.49	5.38 ± 0.79	846.3 ± 27.0	157.26 ± 23.73	40.3 ± 0.2	—	—
WT	1.70	4.26 ± 0.43	262.8 ± 6.5	61.64 ± 6.46	—	44.1 ± 1.1	—
WT	1.64	4.03 ± 0.20	328.2 ± 4.1	81.52 ± 4.09	—	—	—
WT	2.68	4.03 ± 1.18	402.2 ± 33.8	99.77 ± 30.31	—	46.1 ± 0.2	—
WT	1.07	9.06 ± 0.57	1,296.8 ± 25.6	143.13 ± 9.46	—	44.2 ± 0.3	—
WT	0.63	1.31 ± 0.27	570.9 ± 26.1	436.73 ± 93.04	40.9 ± 0.1	—	—
WT	0.84	1.54 ± 0.33	495.5 ± 22.9	320.80 ± 69.26	—	—	—
WT	1.06	2.12 ± 0.33	387.5 ± 13.9	182.82 ± 29.27	—	—	—
WT	0.66	1.40 ± 0.32	568.7 ± 28.1	405.98 ± 95.37	—	—	—
WT	0.89	2.14 ± 0.40	509.9 ± 19.7	238.38 ± 45.67	—	—	—
WT	0.65	4.12 ± 0.70	531.1 ± 22.5	128.95 ± 22.44	—	—	—
WT	0.92	3.27 ± 0.46	419.2 ± 14.7	128.05 ± 18.73	—	—	—
WT	0.73	1.54 ± 0.28	507.3 ± 20.0	329.49 ± 60.53	—	—	—
WT	1.05	3.63 ± 0.25	308.5 ± 5.4	84.97 ± 6.12	—	—	—
WT	0.89	4.89 ± 0.49	308.8 ± 8.9	63.21 ± 6.61	—	—	—
WT	1.09	2.98 ± 0.46	325.9 ± 11.9	109.41 ± 17.41	—	—	—
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
WT	0.88	1.42 ± 0.24	552.2 ± 20.3	387.90 ± 67.43	—	—	—
WT	3.87	5.36 ± 0.73	282.4 ± 9.9	52.70 ± 7.43	—	—	—
WT	*	—	—	—	42.6 ± 0.2	—	—
WT	*	—	—	—	41.3 ± 0.3	—	—
WT	*	—	—	—	42.2 ± 0.1	—	—
WT	*	—	—	—	40.9 ± 0.4	—	—
WT	*	—	—	—	42.0 ± 0.4	—	—
WT	*	—	—	—	42.3 ± 0.2	—	—
WT	*	—	—	—	41.0 ± 0.3	—	—
WT	0.73	4.21 ± 0.68	207.2 ± 9.3	49.21 ± 8.25	38.5 ± 0.4	—	—
WT	6.00	0.97 ± 0.14	174.5 ± 5.1	180.29 ± 26.24	39.2 ± 2.0	—	—
WT	3.18	1.07 ± 0.14	196.6 ± 5.4	184.57 ± 25.05	39.5 ± 0.3	39.5 ± 0.3	—
WT	3.95	4.60 ± 0.17	204.2 ± 2.0	44.41 ± 1.70	40.0 ± 0.2	40.0 ± 0.2	—
WT	5.92	8.42 ± 1.04	267.2 ± 9.9	31.73 ± 4.11	39.7 ± 0.4	39.7 ± 0.4	—
WT	6.66	5.69 ± 0.40	241.9 ± 4.6	42.53 ± 3.09	39.7 ± 0.6	39.7 ± 0.6	—
WT	*	—	—	—	43.4 ± 0.3	—	—
WT	*	—	—	—	41.5 ± 0.3	—	—
WT	*	—	—	—	41.4 ± 0.4	—	—
WT	3.32	3.28 ± 0.33	343.7 ± 8.6	104.86 ± 10.97	40.6 ± 0.2	—	—
WT	3.32	3.73 ± 0.42	348.3 ± 10.0	93.45 ± 10.95	40.3 ± 0.2	—	—
WT	5.02	6.28 ± 0.41	484.2 ± 9.5	77.09 ± 5.25	—	50.8 ± 0.6	—
WT	6.19	4.93 ± 0.32	353.7 ± 6.5	71.80 ± 4.78	—	50.5 ± 1.0	—
WT	4.78	5.21 ± 1.26	376.1 ± 24.4	72.18 ± 18.08	—	49.2 ± 0.3	—
WT	3.86	3.78 ± 0.27	401.2 ± 7.2	106.13 ± 7.72	—	49.1 ± 0.0	—
WT	4.17	4.98 ± 0.56	851.4 ± 25.6	171.02 ± 19.96	—	46.7 ± 0.3	—
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
WT	0.86	6.06 ± 0.43	914.2 ± 18.0	150.78 ± 11.07	39.2 ± 0.4	—	—
WT	1.63	3.40 ± 0.31	604.3 ± 13.8	177.89 ± 16.85	—	—	—
WT	1.63	2.98 ± 0.32	574.1 ± 15.0	192.81 ± 21.30	—	—	—
WT	1.21	1.97 ± 0.26	1,175.1 ± 33.4	597.09 ± 80.47	41.0 ± 0.6	—	—
WT	0.63	2.84 ± 0.32	1,574.0 ± 42.8	554.73 ± 64.89	—	—	—
WT	0.65	1.55 ± 0.18	1,355.7 ± 41.2	872.65 ± 103.27	40.9 ± 0.5	—	—
WT	*	—	—	—	40.9 ± 0.1	—	—
WT	*	—	—	—	39.8 ± 0.3	—	—
WT	1.52	2.87 ± 0.36	702.3 ± 20.5	244.87 ± 31.48	40.7 ± 0.2	—	—
WT	1.37	3.26 ± 0.28	986.6 ± 21.1	302.28 ± 26.99	40.8 ± 0.2	—	—
WT	0.89	2.26 ± 0.29	1,092.0 ± 32.4	483.12 ± 63.41	41.2 ± 0.3	—	—
WT	2.27	2.39 ± 0.29	227.8 ± 6.5	95.49 ± 11.93	—	44.2 ± 0.1	—
WT	2.02	11.37 ± 1.33	482.4 ± 18.1	42.42 ± 5.22	—	46.1 ± 0.2	—
WT	2.23	1.37 ± 0.34	670.7 ± 37.8	489.52 ± 125.00	—	45.6 ± 0.1	—
WT	3.35	5.89 ± 0.34	377.8 ± 6.1	64.11 ± 3.87	—	44.4 ± 0.2	—
WT	3.35	7.36 ± 0.57	383.2 ± 8.6	52.09 ± 4.20	—	44.4 ± 0.2	—
WT	3.35	6.34 ± 0.54	399.0 ± 9.4	62.94 ± 5.54	—	44.4 ± 0.2	—
WT	5.14	1.18 ± 0.21	195.3 ± 7.6	164.85 ± 30.30	—	44.5 ± 0.1	—
WT	2.14	1.31 ± 0.16	252.1 ± 7.9	192.33 ± 24.89	—	45.1 ± 0.2	—
WT	4.71	2.82 ± 0.30	209.7 ± 6.0	74.30 ± 8.06	—	43.9 ± 0.2	—
WT	3.25	4.33 ± 0.43	190.5 ± 4.9	44.01 ± 4.52	39.8 ± 0.2	—	—
WT	2.38	4.00 ± 0.41	264.6 ± 7.0	66.16 ± 7.02	39.7 ± 0.3	—	—
WT	3.22	2.37 ± 0.25	223.4 ± 5.6	94.17 ± 10.29	40.2 ± 0.2	—	—
WT	3.22	2.38 ± 0.25	223.3 ± 5.5	93.87 ± 10.07	40.2 ± 0.2	—	—
WT	3.25	4.33 ± 0.43	190.5 ± 4.9	44.01 ± 4.52	39.8 ± 0.2	—	—
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
WT	2.38	4.00 ± 0.41	264.6 ± 7.0	66.16 ± 7.02	39.7 ± 0.3	—	—
WT	3.22	2.37 ± 0.25	223.4 ± 5.6	94.17 ± 10.29	40.2 ± 0.2	—	—
WT	2.38	4.00 ± 0.41	264.6 ± 7.0	66.16 ± 7.02	39.7 ± 0.3	—	—
WT	—	4.33 ± 0.43	190.5 ± 4.9	44.01 ± 4.52	39.8 ± 0.2	—	—
WT	2.68	3.12 ± 0.19	264.1 ± 4.0	84.56 ± 5.32	—	44.1 ± 0.2	—
WT	*	5.18 ± 0.45	285.8 ± 6.7	55.19 ± 5.01	—	44.0 ± 0.1	—
WT	3.05	5.57 ± 0.75	370.3 ± 14.7	66.45 ± 9.35	—	45.4 ± 0.2	—
WT	2.68	3.10 ± 0.90	256.8 ± 17.2	82.95 ± 24.77	—	45.5 ± 0.4	—
WT	1.56	6.75 ± 0.58	372.3 ± 9.1	55.17 ± 4.94	—	45.1 ± 0.3	—
WT	1.56	6.03 ± 0.60	364.8 ± 10.1	60.49 ± 6.27	—	45.1 ± 0.3	—
WT	1.56	6.71 ± 0.49	374.2 ± 7.7	55.76 ± 4.20	—	45.1 ± 0.3	—
WT	1.72	4.86 ± 0.26	278.3 ± 4.3	57.31 ± 3.22	—	44.2 ± 0.0	—
WT	2.30	4.10 ± 0.31	277.2 ± 6.0	67.61 ± 5.30	—	44.2 ± 0.1	—
WT	1.58	5.07 ± 0.23	284.5 ± 3.9	56.11 ± 2.70	—	44.2 ± 0.0	—
WT	1.73	5.06 ± 0.31	336.4 ± 5.5	66.46 ± 4.20	—	44.3 ± 0.0	—
WT	2.00	7.44 ± 0.89	623.2 ± 21.6	83.78 ± 10.41	—	45.0 ± 0.5	—
WT	2.13	5.42 ± 0.63	804.6 ± 28.9	148.53 ± 18.18	—	46.2 ± 0.2	—
WT	1.40	5.17 ± 0.38	344.9 ± 6.9	66.68 ± 5.11	—	45.5 ± 0.1	—
WT	1.40	6.84 ± 0.46	363.5 ± 7.0	53.16 ± 3.74	—	45.5 ± 0.1	—
WT	1.40	6.86 ± 0.40	358.9 ± 6.0	52.31 ± 3.18	—	45.5 ± 0.1	—
WT	2.10	3.74 ± 0.94	363.9 ± 22.9	97.21 ± 25.06	—	44.5 ± 0.1	—
WT	7.57	4.20 ± 0.42	349.9 ± 9.1	83.40 ± 8.63	—	45.7 ± 0.1	—
WT	4.30	5.00 ± 0.62	324.0 ± 10.7	64.81 ± 8.29	—	43.7 ± 0.1	—
WT	2.09	4.26 ± 0.45	300.9 ± 8.2	70.65 ± 7.72	—	45.6 ± 0.3	—
WT	1.52	4.27 ± 0.31	262.9 ± 4.9	61.63 ± 4.60	—	44.4 ± 0.0	—
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
WT	1.59	2.38 ± 0.47	215.5 ± 9.9	90.71 ± 18.45	—	44.3 ± 0.0	—
WT	3.65	4.72 ± 0.90	353.3 ± 17.5	74.82 ± 14.75	—	46.0 ± 0.1	—
WT	6.46	4.03 ± 0.37	340.5 ± 8.1	84.49 ± 8.09	40.4 ± 0.2	—	—
WT	1.28	1.96 ± 0.24	899.0 ± 28.6	459.05 ± 58.84	40.9 ± 0.2	—	—
WT	1.35	3.21 ± 0.25	1,319.6 ± 24.9	411.12 ± 32.64	39.8 ± 0.2	—	—
WT	0.82	3.48 ± 0.33	1,310.8 ± 31.0	376.33 ± 36.70	40.5 ± 0.5	—	—
WT	1.59	3.75 ± 0.26	1,109.3 ± 19.5	295.93 ± 21.21	40.2 ± 0.2	—	—
WT	0.65	2.74 ± 0.84	1,622.3 ± 97.3	592.54 ± 185.23	39.7 ± 0.3	—	—
WT	1.07	1.68 ± 0.16	607.6 ± 12.9	361.74 ± 35.40	—	—	—
WT	1.21	3.16 ± 0.26	768.3 ± 14.9	242.99 ± 20.37	40.8 ± 0.2	—	—
WT	0.95	3.60 ± 0.39	1,326.6 ± 36.1	368.30 ± 41.18	40.3 ± 0.1	—	—
WT	0.91	2.54 ± 0.41	899.9 ± 34.1	354.62 ± 58.34	40.6 ± 0.2	—	—
WT	5.07	3.39 ± 0.15	91.8 ± 1.0	27.04 ± 1.24	39.8 ± 0.4	—	—
WT	6.75	6.07 ± 0.38	192.9 ± 3.3	31.76 ± 2.04	39.6 ± 0.2	—	—
WT	6.24	5.16 ± 0.43	262.7 ± 5.9	50.90 ± 4.39	40.7 ± 0.3	—	—
WT	2.62	2.71 ± 0.29	455.4 ± 11.5	168.11 ± 18.22	39.8 ± 0.1	—	—
WT	2.59	5.91 ± 0.45	348.1 ± 7.4	58.88 ± 4.69	—	45.0 ± 0.2	—
WT	1.61	4.07 ± 0.22	524.7 ± 8.5	128.79 ± 7.31	—	44.4 ± 0.1	—
WT	3.81	2.61 ± 0.45	188.9 ± 7.7	72.40 ± 12.74	—	47.1 ± 0.5	—
WT	0.74	3.52 ± 0.25	1,050.7 ± 17.9	298.54 ± 21.48	—	44.8 ± 0.0	—
WT	1.43	2.16 ± 0.26	209.4 ± 6.1	96.82 ± 11.97	—	44.7 ± 0.2	—
WT	1.45	3.61 ± 0.27	279.2 ± 5.2	77.37 ± 5.97	—	44.4 ± 0.0	—
W11F	0.71	5.33 ± 0.40	836.9 ± 16.4	156.97 ± 12.12	—	—	—	³
G12N	—	—	—	—	—	—	—	²
T13K	*	—	—	—	—	—	36.0
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
T13K	0.69	0.95 ± 0.39	0.3 ± 0.0	0.36 ± 0.15	—	46.2 ± 2.1	36.0
T13S	1.51	2.95 ± 0.33	408.5 ± 11.1	138.60 ± 15.98	—	46.3 ± 0.6	2.8
T13S	0.10	2.58 ± 0.15	411.4 ± 6.8	159.17 ± 9.35	—	—	2.8
T13S	0.66	31.47 ± 2.04	247.4 ± 6.7	7.86 ± 0.55	39.0 ± 0.6	—	—
T13S	0.94	2.88 ± 0.18	234.6 ± 4.5	81.50 ± 5.41	—	43.8 ± 0.1	2.8
T13S	1.37	3.09 ± 0.15	258.2 ± 4.4	83.65 ± 4.43	—	43.7 ± 0.0	—
S14A	*	8.25 ± 1.02	261.9 ± 8.7	31.82 ± 4.11	40.2 ± 0.3	44.1 ± 0.0	—	²
S14A	1.69	2.88 ± 0.47	177.5 ± 7.1	61.55 ± 10.42	—	43.4 ± 0.1	1.8
T15A	*	5.30 ± 0.48	504.4 ± 13.0	95.25 ± 8.88	39.2 ± 0.2	42.0 ± 0.0	—	²
T15A	*	—	—	—	37.3 ± 0.5	—	9.3
S16A	*	14.01 ± 0.40	125.6 ± 0.0	8.88 ± 0.22	39.4 ± 0.3	43.9 ± 0.0	—	²
S16N	—	—	—	—	—	—	—	²
S17A	*	18.45 ± 3.72	694.9 ± 62.8	37.67 ± 8.23	41.7 ± 0.1	—	—	²
S17A	3.96	6.51 ± 0.50	196.8 ± 4.3	30.23 ± 2.42	38.7 ± 981,798.0	—	—
S17E	*	7.32 ± 0.38	526.1 ± 6.5	71.87 ± 3.90	39.5 ± 0.1	—	—	²
Y18A	*	31.55 ± 3.61	162.4 ± 6.5	5.20 ± 0.65	38.3 ± 0.2	40.6 ± 0.0	—	²
Q19A	*	—	—	—	—	—	—	²
Q19A	1.25	1.68 ± 1.14	0.1 ± 0.0	0.08 ± 0.05	35.2 ± 0.7	—	-1.3
Q19C	*	—	—	—	—	—	—	²
Q19D	0.57	—	—	—	—	—	—
Q19M	0.43	—	—	—	—	46.4 ± 1.5	-18.7
Q19M	*	—	—	—	—	—	—
Q19P	—	—	—	—	—	—	—	²
Q19S	*	—	—	—	36.8 ± 0.2	43.1 ± 0.0	—	²
Q19T	2.19	1.74 ± 0.83	0.1 ± 0.0	0.06 ± 0.03	—	—	-0.4
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
Q19T	*	—	—	—	—	—	-0.2
Q19T	0.89	5.01 ± 0.35	240.1 ± 4.4	47.96 ± 3.43	—	42.8 ± 0.0	-0.2
I20N	0.31	19.41 ± 4.87	44.5 ± 4.0	2.29 ± 0.61	—	—	—	³
E21C	—	—	—	—	—	—	5.4
E21D	0.41	4.55 ± 0.53	32.0 ± 1.0	7.04 ± 0.86	37.5 ± 0.3	37.5 ± 0.3	4.4
E21F	—	—	—	—	—	—	32.3
E21G	—	—	—	—	—	—	8.0
E21H	—	—	—	—	—	—	22.3
E21I	—	—	—	—	—	—	32.6
E21K	—	—	—	—	—	—	27.7
E21N	—	—	—	—	—	—	6.4
E21R	—	—	—	—	—	—	16.9
E21T	—	—	—	—	—	—	6.5
E21W	—	—	—	—	—	—	383.6
E21Y	4.45	1.52 ± 0.20	184.4 ± 5.4	120.95 ± 16.52	40.3 ± 0.3	40.3 ± 0.3	27.1
G23A	0.73	1.41 ± 0.23	619.2 ± 22.3	440.37 ± 74.87	40.1 ± 0.2	—	1.8
G23A	1.99	1.31 ± 0.30	269.2 ± 11.3	205.91 ± 47.41	—	45.0 ± 0.0	1.8
D25Q	0.64	4.21 ± 0.35	406.3 ± 8.7	96.43 ± 8.28	40.3 ± 0.1	—	—
D25Q	6.91	0.85 ± 0.09	66.3 ± 1.7	77.95 ± 8.88	—	—	—
D25Q	0.64	4.21 ± 0.35	406.3 ± 8.7	96.43 ± 8.28	—	—	—
D25Q	5.72	4.53 ± 1.15	352.8 ± 23.5	77.93 ± 20.51	—	45.5 ± 0.0	—
E26K	0.62	6.81 ± 0.48	638.8 ± 12.9	93.81 ± 6.93	38.4 ± 0.3	—	-0.9
S32L	—	—	—	—	—	—	—	²
W34A	—	—	—	—	—	—	—	²
D35E	2.00	2.72 ± 0.31	16.3 ± 0.4	5.97 ± 0.70	—	—	11.6
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
D35W	0.46	2.80 ± 0.55	13.0 ± 0.6	4.66 ± 0.94	—	—	14.7
D35W	0.34	2.45 ± 1.03	2.0 ± 0.2	0.83 ± 0.35	—	46.8 ± 3.6	14.0
T36I	—	—	—	—	—	—	—
T36N	5.50	6.57 ± 1.21	23.9 ± 1.2	3.63 ± 0.69	—	46.8 ± 0.1	-0.2
F37N	0.26	4.88 ± 0.15	8.3 ± 0.1	1.70 ± 0.06	—	—	—	³
C38S	1.03	6.63 ± 0.64	1,100.6 ± 25.8	165.92 ± 16.39	40.3 ± 0.3	—	1.1
C38S	1.30	10.95 ± 1.34	570.7 ± 22.3	52.14 ± 6.71	41.6 ± 0.4	—	1.1
C38S	0.75	3.58 ± 0.33	1,340.5 ± 28.8	374.51 ± 34.98	41.5 ± 0.1	—	1.1
C38S	1.12	2.67 ± 0.37	214.7 ± 7.2	80.46 ± 11.59	—	42.2 ± 0.2	-1.4
I40T	1.41	4.05 ± 0.21	265.2 ± 3.8	65.54 ± 3.50	—	43.3 ± 0.2	2.2
P41L	*	—	—	—	—	—	—
G46C	0.04	40.81 ± 25.63	4.4 ± 1.2	0.11 ± 0.07	—	—	—	³
C49S	0.85	6.63 ± 3.19	570.0 ± 74.3	86.02 ± 42.89	40.0 ± 0.0	—	—
V52A	2.06	5.30 ± 0.91	107.2 ± 5.0	20.22 ± 3.61	—	40.9 ± 0.2	—
V52G	*	8.25 ± 0.54	562.9 ± 10.8	68.19 ± 4.55	39.3 ± 0.1	—	—	²
F57Y	2.75	5.11 ± 0.60	37.3 ± 1.1	7.29 ± 0.88	—	45.2 ± 0.1	-0.1
F57Y	1.40	4.18 ± 0.37	309.1 ± 6.9	74.01 ± 6.70	—	45.6 ± 0.0	-0.1
H58R	1.22	5.64 ± 0.68	665.5 ± 21.2	118.04 ± 14.68	—	—	—	³
H59R	1.35	4.83 ± 0.35	576.2 ± 10.7	119.33 ± 8.87	—	—	—	³
H59R	4.78	9.79 ± 0.80	15.3 ± 0.4	1.56 ± 0.13	—	52.6 ± 0.4	-0.2
H59R	1.53	3.09 ± 0.20	315.6 ± 5.3	102.28 ± 6.82	—	47.0 ± 0.0	-0.3
K61A	1.55	3.79 ± 0.40	167.6 ± 4.4	44.28 ± 4.78	38.7 ± 0.4	—	1.3
K61C	2.86	1.94 ± 0.26	111.8 ± 3.7	57.63 ± 8.08	38.4 ± 0.2	—	2.4
K61C	2.87	1.56 ± 0.19	119.8 ± 3.0	76.81 ± 9.43	37.8 ± 0.3	—	2.4
K61D	1.11	3.96 ± 0.39	116.3 ± 2.8	29.36 ± 2.96	38.3 ± 0.3	—	5.6
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
K61E	1.36	3.79 ± 0.28	141.6 ± 2.7	37.39 ± 2.87	38.8 ± 0.2	—	0.8
K61F	3.29	3.66 ± 0.28	21.3 ± 0.4	5.81 ± 0.46	38.4 ± 0.6	—	7.6
K61F	1.87	2.91 ± 0.21	120.6 ± 2.2	41.43 ± 3.15	36.4 ± 0.4	—	7.6
K61G	1.81	4.07 ± 0.24	194.4 ± 2.9	47.72 ± 2.85	37.8 ± 0.7	—	4.6
K61G	0.90	1.96 ± 0.17	10.3 ± 0.2	5.25 ± 0.47	38.3 ± 0.4	—	—
K61H	6.16	1.95 ± 0.19	164.0 ± 3.6	84.15 ± 8.28	38.2 ± 0.1	—	3.3
K61I	1.80	2.43 ± 0.19	104.7 ± 2.0	43.13 ± 3.46	38.4 ± 0.2	—	1.1
K61L	0.83	2.37 ± 0.30	60.7 ± 1.9	25.68 ± 3.33	40.6 ± 0.8	—	5.5
K61M	4.60	4.59 ± 0.53	200.1 ± 6.0	43.57 ± 5.17	38.0 ± 0.1	—	2.6
K61N	3.72	4.20 ± 0.51	184.7 ± 5.8	43.95 ± 5.51	33.5 ± 10.2	—	3.1
K61N	3.29	4.11 ± 0.28	129.1 ± 2.2	31.41 ± 2.18	37.7 ± 0.3	—	3.1
K61P	1.49	2.90 ± 0.26	81.6 ± 1.8	28.19 ± 2.62	38.9 ± 0.3	—	11.7
K61Q	1.42	4.28 ± 1.38	137.0 ± 12.5	31.98 ± 10.68	39.3 ± 0.2	—	0.8
K61R	2.60	2.82 ± 0.25	109.8 ± 2.3	38.93 ± 3.50	39.2 ± 0.3	—	1.4
K61S	0.45	4.32 ± 0.84	142.2 ± 7.2	32.91 ± 6.62	—	46.2 ± 0.3	0.0
K61S	3.21	1.17 ± 0.06	147.7 ± 1.9	126.25 ± 7.19	38.5 ± 0.2	—	2.1
K61S	3.07	4.17 ± 0.48	308.2 ± 9.1	73.90 ± 8.78	—	46.3 ± 0.3	2.1
K61W	1.94	3.95 ± 0.43	74.2 ± 2.2	18.78 ± 2.13	38.2 ± 0.7	—	7.8
K61Y	0.47	1.86 ± 0.17	93.3 ± 1.9	50.18 ± 4.73	38.0 ± 0.5	—	7.7
D63A	0.01	—	—	0.33 ± 0.09	—	—	0.0
D63C	0.98	0.69 ± 1.31	0.0 ± 0.0	0.07 ± 0.14	—	—	—
D63F	0.77	4.13 ± 9.33	0.1 ± 0.0	0.01 ± 0.03	—	—	—
D63M	0.10	0.24 ± 1.13	0.2 ± 0.1	0.69 ± 3.33	—	—	—
D63M	0.62	0.03 ± 0.16	0.1 ± 0.0	3.23 ± 19.66	—	—	—
D63N	0.44	0.28 ± 0.43	0.2 ± 0.0	0.56 ± 0.87	—	—	—
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
D63P	0.28	—	—	—	—	—	—
D63R	0.13	—	—	0.02 ± 0.00	—	—	—
D63S	0.35	0.09 ± 0.16	0.1 ± 0.0	1.39 ± 2.61	—	—	—
K68H	2.79	3.83 ± 0.38	397.6 ± 10.1	103.74 ± 10.74	—	46.8 ± 1.1	2.4
K68H	3.73	4.20 ± 1.14	255.3 ± 17.4	60.76 ± 16.94	—	45.3 ± 0.1	—
F72A	*	5.47 ± 0.28	502.2 ± 6.5	91.79 ± 4.76	38.6 ± 0.4	—	—	²
Y75F	0.11	0.44 ± 0.49	1.5 ± 0.3	3.51 ± 3.96	40.1 ± 1.7	—	2.0
Y75F	3.87	6.57 ± 1.96	251.7 ± 22.6	38.30 ± 11.92	—	46.4 ± 0.6	2.0
Y75N	0.20	7.88 ± 0.55	410.7 ± 8.0	52.13 ± 3.77	—	—	—	³
Y75N	0.09	2.08 ± 0.36	318.1 ± 14.9	153.23 ± 27.75	38.0 ± 1.2	—	10.8
R76A	—	—	—	—	—	—	—	²
R76L	0.54	43.36 ± 1.82	7.4 ± 0.1	0.17 ± 0.01	—	—	—	³
R76Q	0.70	6.62 ± 2.33	0.8 ± 0.1	0.13 ± 0.05	—	—	-0.1
F77D	0.22	—	—	—	—	—	—
F77W	0.38	73.32 ± 94.87	20.0 ± 14.3	0.27 ± 0.40	—	—	—
S78A	3.12	20.08 ± 1.37	190.0 ± 4.9	9.46 ± 0.69	—	37.5 ± 0.1	1.9
V79I	3.63	3.04 ± 0.35	493.6 ± 12.3	162.30 ± 18.85	39.2 ± 0.2	—	—
V79I	3.48	3.38 ± 0.28	523.3 ± 10.7	154.71 ± 13.07	—	—	—
V79T	2.67	5.29 ± 2.61	239.4 ± 31.8	45.23 ± 23.12	—	46.7 ± 0.1	4.7
A80P	—	—	—	—	—	—	—
A80P	0.30	0.17 ± 0.11	0.5 ± 0.1	2.94 ± 1.92	—	30.6 ± 10.1	190.9
A80P	0.38	8.09 ± 0.55	28.2 ± 0.6	3.48 ± 0.25	34.9 ± 0.5	—	—
A80S	2.04	3.23 ± 0.30	404.0 ± 9.3	125.15 ± 12.09	—	43.4 ± 0.1	2.8
A80S	0.66	13.08 ± 1.18	2,139.3 ± 64.0	163.52 ± 15.56	38.8 ± 0.6	38.8 ± 0.6	2.8
A80S	2.40	3.44 ± 0.27	514.9 ± 10.0	149.68 ± 12.09	42.2 ± 0.2	—	2.7
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
A80S	2.31	1.64 ± 0.29	78.1 ± 3.7	47.64 ± 8.67	—	46.6 ± 0.2	2.7
A80S	3.19	1.86 ± 0.29	349.3 ± 12.2	187.42 ± 29.76	40.8 ± 0.2	—	1.7
A88E	2.14	0.57 ± 0.14	249.7 ± 11.6	436.07 ± 107.07	—	44.3 ± 0.0	-1.4
A88E	2.29	6.89 ± 0.87	367.4 ± 13.3	53.31 ± 7.04	—	44.5 ± 0.2	—
A88E	3.00	4.15 ± 0.53	335.8 ± 11.0	80.89 ± 10.64	—	48.1 ± 0.3	-1.4
A88K	0.79	4.46 ± 0.60	307.4 ± 10.5	68.93 ± 9.53	—	—	—	³
I90V	1.25	6.67 ± 0.28	790.7 ± 9.2	118.48 ± 5.19	—	—	—	³
I91A	0.73	9.83 ± 3.63	202.7 ± 22.3	20.62 ± 7.94	37.6 ± 431,281.0	—	—
I91A	0.11	5.06 ± 0.37	1,005.1 ± 20.9	198.84 ± 14.97	39.4 ± 0.1	—	-2.8
I91C	5.45	3.58 ± 0.15	19.6 ± 0.2	5.47 ± 0.24	38.1 ± 0.2	—	—
I91D	0.72	11.21 ± 0.69	89.6 ± 1.8	7.99 ± 0.51	37.4 ± 183,653.0	—	—
I91E	*	6.71 ± 0.79	692.7 ± 28.1	103.26 ± 12.77	39.7 ± 0.3	—	—	²
I91G	0.45	9.30 ± 3.31	93.7 ± 10.2	10.08 ± 3.75	—	—	—
I91H	3.69	11.59 ± 0.81	181.2 ± 4.1	15.64 ± 1.15	37.6 ± 221,464.0	—	—
I91K	0.31	3.73 ± 0.92	4,015.9 ± 262.1	1,075.53 ± 275.42	39.7 ± 0.3	—	—
I91M	0.87	9.04 ± 0.55	148.5 ± 2.8	16.43 ± 1.05	37.5 ± 0.3	—	—
I91N	1.51	9.39 ± 0.98	183.9 ± 5.9	19.59 ± 2.14	36.0 ± 0.2	—	—
I91Q	3.47	13.34 ± 0.58	209.0 ± 3.0	15.66 ± 0.72	37.8 ± 2,701,870.0	—	—
I91R	0.90	8.45 ± 2.50	207.6 ± 18.4	24.56 ± 7.59	36.2 ± 0.2	—	—
I91R	0.16	6.31 ± 0.36	271.9 ± 4.3	43.08 ± 2.53	39.1 ± 0.2	—	-8.0
I91S	1.40	10.13 ± 3.43	299.2 ± 29.0	29.54 ± 10.41	36.8 ± 0.2	—	—
I91V	1.88	10.44 ± 1.37	22.5 ± 0.9	2.16 ± 0.30	37.9 ± 88,473.6	—	—
I91Y	2.71	10.90 ± 0.60	248.8 ± 4.3	22.81 ± 1.31	35.8 ± 0.2	—	—
H101R	*	10.62 ± 0.53	868.1 ± 13.0	81.83 ± 4.33	40.1 ± 0.1	48.2 ± 0.0	—	²
I106A	1.03	5.15 ± 0.34	732.2 ± 13.0	142.12 ± 9.74	—	—	—
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
L108E	3.70	4.31 ± 0.22	404.9 ± 5.4	93.91 ± 4.95	—	—	-1.8
L108E	1.84	8.47 ± 1.13	370.1 ± 14.7	43.67 ± 6.05	—	43.3 ± 0.0	0.8
L108M	2.35	4.14 ± 0.35	391.2 ± 8.6	94.54 ± 8.36	—	47.3 ± 0.1	0.6
L108N	2.91	4.27 ± 0.67	330.4 ± 13.4	77.38 ± 12.49	—	44.8 ± 0.0	0.0
L108S	0.26	2.76 ± 0.50	95.4 ± 4.1	34.55 ± 6.39	38.1 ± 0.5	—	-1.1
M114S	1.18	13.79 ± 1.07	257.2 ± 6.8	18.65 ± 1.53	—	—	5.0
M114S	0.68	5.80 ± 0.50	48.9 ± 1.2	8.42 ± 0.76	—	40.0 ± 34.1	5.0
T116I	3.42	29.39 ± 2.92	261.6 ± 10.7	8.90 ± 0.95	—	42.6 ± 0.5	-7.3
T116I	1.64	21.76 ± 14.44	529.5 ± 133.5	24.34 ± 17.28	—	43.7 ± 0.0	15.7
T116I	0.90	13.87 ± 1.79	140.3 ± 6.1	10.11 ± 1.38	—	44.1 ± 0.1	—
T116S	1.61	2.21 ± 0.44	138.1 ± 6.3	62.58 ± 12.74	—	43.1 ± 0.1	4.0
Y118A	0.31	11.90 ± 1.18	11.2 ± 0.4	0.94 ± 0.10	—	46.0 ± 0.1	—
Y118F	3.73	0.87 ± 0.86	108.4 ± 20.7	124.90 ± 126.20	—	44.5 ± 0.6	3.5
Y118F	0.34	—	—	—	—	—	3.0
H119A	*	15.10 ± 3.36	116.9 ± 8.7	7.79 ± 1.73	40.2 ± 0.2	—	—	²
H119D	0.61	—	—	0.00 ± 0.00	—	—	1.6
H119D	0.37	—	—	4.42 ± 0.41	—	—	1.1
H119D	0.19	—	—	0.03 ± 0.00	—	—	2.8
H119D	—	—	—	—	—	—	10.1
H119E	—	—	—	—	—	—	—	²
H119M	0.55	17.02 ± 4.07	9.8 ± 0.8	0.57 ± 0.15	—	—	—
H119N	*	23.22 ± 2.20	2.2 ± 0.0	—	40.3 ± 0.6	—	—	²
H119Q	0.80	67.26 ± 12.70	153.1 ± 16.0	2.28 ± 0.49	—	45.7 ± 0.6	—
H119T	0.84	66.09 ± 31.10	20.7 ± 5.2	0.31 ± 0.17	—	—	—	³
H119T	1.34	—	—	0.24 ± 0.01	—	45.1 ± 0.5	3.1
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
H119V	0.43	44.36 ± 5.88	5.2 ± 0.3	0.12 ± 0.02	—	—	—
W120A	—	—	—	—	—	—	—	²
W120D	0.18	12.34 ± 0.44	47.0 ± 0.5	3.81 ± 0.14	—	—	—	³
W120D	*	15.53 ± 6.04	140.8 ± 17.8	9.07 ± 3.71	35.6 ± 0.0	—	5.9
W120D	0.24	—	—	0.06 ± 0.00	—	—	5.9
W120E	0.11	2.02 ± 0.76	8.9 ± 0.6	4.43 ± 1.69	40.9 ± 0.1	—	6.5
W120E	0.93	—	—	0.07 ± 0.00	—	44.8 ± 0.3	4.7
W120F	*	16.08 ± 2.07	387.5 ± 17.3	24.03 ± 3.25	41.7 ± 0.4	50.7 ± 0.0	—	²
W120G	0.38	—	—	0.12 ± 0.00	—	—	—	³
W120H	*	89.18 ± 4.31	69.3 ± 2.2	0.87 ± 0.00	40.1 ± 0.1	46.2 ± 0.0	—	²
W120K	0.25	56.50 ± 10.55	1.7 ± 0.2	0.03 ± 0.01	—	—	—	³
W120L	1.43	—	—	0.61 ± 0.02	37.6 ± 633,161.0	—	—
W120M	1.46	50.86 ± 3.20	100.2 ± 3.2	1.97 ± 0.14	—	43.9 ± 0.1	5.2
W120N	0.29	22.72 ± 11.58	2.4 ± 0.5	0.11 ± 0.06	—	—	—
W120N	0.89	0.71 ± 0.29	3.6 ± 0.3	5.01 ± 2.11	—	48.2 ± 0.1	4.6
W120P	—	—	—	—	—	—	—
W120Q	0.63	—	—	0.29 ± 0.02	—	—	—	³
W120S	0.74	13.51 ± 2.65	1.2 ± 0.1	0.09 ± 0.02	—	—	0.0
W120S	0.38	—	—	0.10 ± 0.00	40.3 ± 0.9	—	—
W120T	0.46	0.37 ± 0.25	0.4 ± 0.0	1.01 ± 0.69	—	47.0 ± 2.1	-0.2
W120Y	4.99	16.94 ± 2.50	7.5 ± 0.4	0.44 ± 0.07	34.8 ± 0.2	—	—
W120Y	2.37	24.12 ± 3.29	1.6 ± 0.1	0.07 ± 0.01	—	50.2 ± 1.2	11.1
D121F	—	—	—	—	—	—	—	²
L122Q	0.68	2.21 ± 0.82	88.1 ± 7.6	39.95 ± 15.23	36.8 ± 0.1	—	7.9
L122Q	0.20	2.72 ± 0.23	812.9 ± 20.2	298.77 ± 26.14	36.3 ± 0.2	—	8.0
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
P123D	—	—	—	—	—	—	21.2
Q124D	0.75	4.60 ± 0.46	193.8 ± 4.9	42.15 ± 4.39	36.2 ± 0.4	—	10.1
Q124G	0.25	6.17 ± 0.27	71.3 ± 0.9	11.55 ± 0.52	36.6 ± 0.2	—	—
Q124I	1.74	3.43 ± 0.22	152.5 ± 2.3	44.49 ± 2.96	40.4 ± 0.7	—	5.4
Q124P	0.75	4.90 ± 0.41	313.4 ± 6.9	64.02 ± 5.48	38.6 ± 0.4	—	336.0
Q124P	1.21	5.10 ± 0.33	214.3 ± 3.7	42.01 ± 2.78	40.4 ± 0.4	—	—
Q124S	0.61	5.66 ± 0.77	141.2 ± 5.3	24.94 ± 3.52	—	—	—
Q124T	1.30	5.65 ± 0.46	181.6 ± 4.0	32.13 ± 2.69	39.7 ± 0.8	—	7.5
Q124T	0.21	5.42 ± 0.52	133.8 ± 3.5	24.67 ± 2.47	36.3 ± 0.2	—	—
W125F	1.60	4.77 ± 0.44	47.6 ± 1.1	9.97 ± 0.96	36.8 ± 0.4	—	6.3
W125R	0.18	3.71 ± 0.99	62.4 ± 4.1	16.81 ± 4.60	41.3 ± 1.5	—	10.1
E127D	0.22	2.42 ± 0.32	136.6 ± 4.3	56.50 ± 7.77	39.2 ± 0.3	—	—
D128E	0.36	5.54 ± 0.38	996.4 ± 18.6	179.93 ± 12.78	39.7 ± 0.1	—	1.7
D128E	0.49	5.66 ± 0.55	154.1 ± 4.0	27.24 ± 2.73	38.3 ± 0.4	—	1.8
D128E	0.20	7.75 ± 1.01	532.6 ± 19.6	68.70 ± 9.31	39.3 ± 0.2	—	1.8
E129Q	0.90	5.17 ± 0.46	348.9 ± 8.3	67.49 ± 6.21	—	43.4 ± 0.0	-1.1
T133Y	3.22	1.58 ± 0.16	248.5 ± 6.2	157.21 ± 15.94	—	44.5 ± 0.2	6.8
Q139E	4.17	6.47 ± 1.12	754.0 ± 35.2	116.47 ± 20.92	—	47.7 ± 1.3	-0.6
Q139K	0.47	6.52 ± 0.52	32.3 ± 0.7	4.96 ± 0.41	—	45.3 ± 0.2	-3.8
Q139K	0.47	6.52 ± 0.51	32.3 ± 0.7	4.96 ± 0.41	—	45.3 ± 0.2	-3.8
Q139K	0.34	5.18 ± 1.16	115.5 ± 6.9	22.31 ± 5.16	—	45.1 ± 0.1	-0.4
Q139K	2.32	3.65 ± 0.27	332.4 ± 6.2	91.14 ± 7.00	—	45.3 ± 0.1	-1.8
Q139K	2.90	2.77 ± 0.34	284.5 ± 8.4	102.78 ± 13.05	—	43.3 ± 0.2	—
Q139K	5.15	5.62 ± 0.76	214.1 ± 7.9	38.07 ± 5.35	—	45.5 ± 0.3	—
K142R	0.54	4.57 ± 1.26	60.2 ± 4.3	13.17 ± 3.74	—	—	—
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
Y144W	1.70	2.48 ± 0.47	280.5 ± 12.4	113.00 ± 22.09	—	43.8 ± 0.4	-8.8
A145C	2.42	4.02 ± 0.28	368.6 ± 6.5	91.74 ± 6.52	—	44.3 ± 0.1	3.0
A145C	—	—	—	—	—	—	—
A145E	2.62	4.95 ± 0.38	295.1 ± 6.0	59.61 ± 4.74	40.8 ± 0.1	—	—
M149I	2.60	9.03 ± 0.70	377.2 ± 8.9	41.79 ± 3.38	—	42.2 ± 0.1	88.5
D150V	0.38	3.02 ± 0.71	1.0 ± 0.1	0.32 ± 0.08	39.6 ± 0.3	—	19.0
D150V	2.28	2.67 ± 0.49	255.9 ± 12.0	95.94 ± 18.08	—	45.1 ± 0.2	6.3
E154A	0.27	30.92 ± 13.46	112.2 ± 20.0	3.63 ± 1.71	—	—	1.6
E154D	*	3.46 ± 0.76	720.9 ± 39.0	208.26 ± 47.19	38.8 ± 0.3	49.5 ± 0.0	—	²
E154F	0.72	6.49 ± 0.56	2,334.7 ± 61.1	359.48 ± 32.42	—	—	—
N157D	0.72	6.32 ± 0.56	330.1 ± 8.2	52.25 ± 4.80	—	47.5 ± 0.0	-1.0
N157H	2.06	2.17 ± 0.46	344.9 ± 16.7	159.00 ± 34.33	—	44.1 ± 0.0	-0.4
N157T	0.26	2.55 ± 0.59	19.7 ± 1.1	7.73 ± 1.83	—	—	—
N157Y	1.15	7.39 ± 0.93	1,508.5 ± 51.8	204.18 ± 26.62	40.8 ± 0.2	—	-1.6
W158N	2.79	4.95 ± 0.18	850.8 ± 7.9	171.78 ± 6.35	—	—	—	³
N160A	3.59	14.26 ± 1.61	402.4 ± 15.5	28.22 ± 3.37	—	44.1 ± 0.4	2.4
N160C	4.07	4.95 ± 0.66	499.7 ± 17.8	101.02 ± 14.02	—	42.9 ± 0.1	—
N160H	0.99	14.81 ± 0.91	144.3 ± 3.0	9.74 ± 0.63	—	41.2 ± 0.0	20.6
N160S	3.61	6.71 ± 0.56	393.5 ± 9.3	58.64 ± 5.07	—	42.5 ± 0.1	—
N160V	1.00	8.66 ± 1.44	913.8 ± 44.3	105.57 ± 18.33	38.0 ± 205,656.0	—	—
N160V	1.85	8.66 ± 1.44	494.0 ± 23.9	57.06 ± 9.91	39.6 ± 0.2	—	—
N160V	2.69	3.18 ± 0.87	121.6 ± 7.7	38.28 ± 10.78	—	—	—
N160V	1.52	3.86 ± 1.02	712.0 ± 41.2	184.63 ± 50.08	39.1 ± 0.3	—	4.2
N163A	*	11.95 ± 0.91	6.5 ± 0.0	0.65 ± 0.00	41.2 ± 0.3	44.4 ± 0.0	—	²
N163C	*	5.42 ± 0.32	21.6 ± 0.0	3.90 ± 0.22	38.1 ± 0.3	40.5 ± 0.0	—	²
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
N163D	*	15.19 ± 1.41	10.8 ± 0.0	0.65 ± 0.00	38.5 ± 0.3	—	—	²
N163E	—	—	—	—	—	—	—	²
N163G	2.38	9.68 ± 0.47	2.4 ± 0.0	0.25 ± 0.01	—	41.7 ± 0.0	6.0
N163H	*	—	—	—	—	43.8 ± 0.1	21.9
N163K	—	—	—	—	—	—	—	²
N163Q	0.11	7.01 ± 4.09	1.1 ± 0.2	0.16 ± 0.10	—	—	—
N163S	0.30	2.67 ± 0.41	1,758.0 ± 62.2	658.99 ± 104.13	38.1 ± 0.5	—	—
N163T	0.17	23.01 ± 2.67	4.8 ± 0.2	0.21 ± 0.03	—	—	—	³
N163T	0.03	—	—	—	—	—	4.5
N163T	0.08	—	—	—	—	—	—
E164A	*	1.01 ± 0.17	—	—	46.0 ± 0.1	53.9 ± 0.0	—	²
E164G	—	—	—	—	—	—	—	²
E164R	—	—	—	—	—	—	—	²
P165A	0.20	3.73 ± 0.33	554.7 ± 12.5	148.77 ± 13.64	39.5 ± 0.2	—	—
Y166F	0.37	5.16 ± 0.67	5.9 ± 0.2	1.14 ± 0.15	—	—	—
Y166F	0.27	4.95 ± 1.46	8.7 ± 0.7	1.75 ± 0.54	—	—	—
Y166H	1.34	22.35 ± 1.57	593.9 ± 16.1	26.57 ± 2.00	—	44.5 ± 0.1	3.4
Y166H	1.28	14.38 ± 0.95	534.3 ± 12.0	37.14 ± 2.59	—	44.1 ± 0.2	3.4
Y166K	0.34	2.24 ± 0.55	1.8 ± 0.1	0.80 ± 0.20	—	48.4 ± 0.5	11.4
Y166P	—	—	—	—	—	—	—	²
Y166S	1.64	6.67 ± 1.04	43.3 ± 1.9	6.49 ± 1.05	—	—	8.2
Y166W	1.90	7.58 ± 0.52	9.7 ± 0.2	1.28 ± 0.09	—	—	—	³
C167A	*	14.56 ± 1.27	391.8 ± 10.8	26.84 ± 2.38	39.8 ± 0.5	51.1 ± 0.0	—	²
C167D	1.60	57.52 ± 10.92	87.6 ± 8.8	1.52 ± 0.33	—	46.4 ± 0.1	7.6
C167E	0.70	5.14 ± 0.20	183.5 ± 1.9	35.68 ± 1.44	—	—	—	³
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
C167H	0.36	3.73 ± 0.63	168.2 ± 6.2	45.12 ± 7.79	—	—	—
C167H	—	—	—	—	—	—	—
C167N	2.33	37.36 ± 2.90	487.4 ± 16.6	13.05 ± 1.11	—	—	—	³
C167N	1.72	1.67 ± 0.57	232.3 ± 17.6	138.82 ± 48.64	—	44.0 ± 0.2	4.1
C167N	0.33	7.09 ± 0.71	438.0 ± 12.5	61.79 ± 6.41	40.2 ± 0.2	—	6.7
C167Q	*	4.92 ± 0.19	413.5 ± 4.3	84.00 ± 3.46	38.9 ± 0.1	48.2 ± 0.0	—	²
C167S	5.13	71.46 ± 9.54	17.0 ± 1.3	0.24 ± 0.04	—	—	3.0
C167S	2.28	—	—	0.12 ± 0.00	—	48.0 ± 0.2	3.0
C167T	0.20	2.06 ± 0.21	344.5 ± 8.4	167.23 ± 17.28	37.0 ± 0.3	—	9.6
C167T	1.34	—	—	0.19 ± 0.01	41.2 ± 0.8	—	12.2
A168N	0.42	7.31 ± 0.40	157.0 ± 2.4	21.46 ± 1.23	—	—	—	³
S169A	1.35	4.33 ± 0.45	515.6 ± 14.8	119.08 ± 12.78	—	44.8 ± 0.5	-2.2
S169D	0.13	68.87 ± 91.89	1.6 ± 1.1	0.02 ± 0.03	—	—	—
S169N	0.28	10.12 ± 5.32	1.6 ± 0.2	0.16 ± 0.09	—	—	—
I170A	0.43	2.41 ± 0.16	253.5 ± 3.9	105.24 ± 7.16	—	—	4.8
I170A	0.37	3.38 ± 0.18	673.5 ± 8.8	199.00 ± 10.77	37.4 ± 0.1	—	4.8
I170Y	0.86	4.82 ± 0.46	432.8 ± 11.0	89.75 ± 8.93	35.0 ± 0.2	—	6.7
L171A	*	11.09 ± 0.42	662.4 ± 6.5	59.75 ± 2.38	38.8 ± 0.5	42.9 ± 0.0	—	²
L171F	2.70	1.59 ± 0.12	261.4 ± 4.4	164.73 ± 12.91	—	47.0 ± 0.0	1.6
L171F	8.12	5.65 ± 0.36	362.9 ± 7.4	64.25 ± 4.29	—	51.5 ± 0.0	—
L171F	5.09	3.00 ± 0.71	63.7 ± 0.1	21.24 ± 5.02	—	51.5 ± 0.0	—
L171H	3.26	7.23 ± 0.81	256.6 ± 8.3	35.48 ± 4.14	—	46.7 ± 0.1	—
L171I	0.69	18.69 ± 3.62	427.8 ± 29.3	22.89 ± 4.70	—	—	—	³
L171I	0.66	5.12 ± 0.23	207.2 ± 2.7	40.50 ± 1.92	—	44.0 ± 0.1	39.3
L171I	1.53	7.84 ± 0.44	334.1 ± 5.4	42.60 ± 2.49	—	43.8 ± 0.0	—
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
L171M	0.79	3.42 ± 0.21	247.9 ± 3.7	72.51 ± 4.49	—	45.5 ± 0.1	—
L171Q	1.12	7.99 ± 0.66	77.1 ± 1.8	9.66 ± 0.83	—	—	—	³
L171Q	1.27	2.75 ± 0.34	554.7 ± 17.3	201.42 ± 25.79	—	43.0 ± 0.1	3.9
L171R	*	3.36 ± 0.23	331.2 ± 6.5	98.50 ± 6.93	38.8 ± 0.3	44.6 ± 0.0	—	²
L171Y	0.91	1.10 ± 0.12	410.4 ± 9.4	373.98 ± 42.07	37.2 ± 0.1	—	—
L171Y	1.57	1.67 ± 0.57	255.1 ± 19.4	152.58 ± 53.53	—	42.2 ± 0.0	-0.4
Y173L	0.17	1.28 ± 0.18	193.7 ± 7.4	151.01 ± 22.19	—	—	-3.6
Y173L	0.03	—	—	—	—	—	10.1
Y173L	0.55	2.90 ± 0.12	97.1 ± 1.2	33.53 ± 1.47	—	—	10.1
T175A	0.38	10.60 ± 0.79	425.0 ± 9.7	40.10 ± 3.13	—	—	—	³
T175C	0.33	14.14 ± 1.21	2,190.6 ± 61.4	154.88 ± 13.95	—	—	—	³
T175H	0.04	4.89 ± 0.90	362.3 ± 17.2	74.03 ± 14.12	37.6 ± 1.0	—	10.0
T175H	0.10	0.20 ± 0.28	36.9 ± 7.3	181.10 ± 251.42	—	—	—
T175K	2.03	6.21 ± 0.54	708.5 ± 16.7	114.15 ± 10.27	—	—	—	³
T175M	2.50	5.04 ± 0.72	283.7 ± 12.1	56.30 ± 8.37	—	43.1 ± 0.1	0.3
T175R	*	3.59 ± 0.15	655.9 ± 6.5	182.71 ± 7.79	38.1 ± 0.1	—	—	²
E177A	*	5.98 ± 0.22	807.5 ± 8.7	135.09 ± 5.20	37.5 ± 0.2	—	—	²
E177A	6.12	6.09 ± 0.58	325.0 ± 8.6	53.40 ± 5.29	—	50.3 ± 0.4	3.1
E177A	1.75	9.41 ± 0.74	261.2 ± 6.3	27.77 ± 2.28	—	43.8 ± 0.4	—
E177C	1.52	6.13 ± 0.26	70.8 ± 0.8	11.55 ± 0.51	—	—	—	³
E177D	1.93	—	—	—	37.9 ± 80,708.3	—	—
E177D	1.97	5.04 ± 0.58	404.1 ± 12.5	80.17 ± 9.60	37.8 ± 76,045.9	—	—
E177I	—	—	—	—	—	—	—
E177K	*	6.19 ± 0.30	454.6 ± 6.5	73.39 ± 3.68	36.7 ± 0.3	—	—	²
E177L	*	7.48 ± 0.36	549.9 ± 8.7	73.60 ± 3.68	39.6 ± 0.4	—	—	²
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
E177Q	0.58	8.17 ± 0.46	799.4 ± 13.0	97.81 ± 5.78	—	—	—	³
E177T	2.73	8.71 ± 0.59	504.9 ± 9.9	57.94 ± 4.08	—	—	—	³
E177T	2.10	6.46 ± 1.08	259.2 ± 12.2	40.14 ± 6.96	—	43.6 ± 0.1	—
E177W	0.39	5.66 ± 0.39	101.3 ± 1.9	17.88 ± 1.29	40.8 ± 0.1	—	5.2
E177W	0.20	14.97 ± 1.83	20.8 ± 0.9	1.39 ± 0.18	38.5 ± 0.4	—	5.2
H178A	*	7.67 ± 0.73	93.1 ± 2.2	12.12 ± 1.08	39.6 ± 0.4	—	—	²
H178C	0.04	0.99 ± 0.32	73.5 ± 5.4	74.43 ± 24.59	—	—	3.5
H178C	0.16	6.81 ± 0.30	113.2 ± 1.4	16.62 ± 0.76	—	—	3.4
H178F	0.12	6.07 ± 0.36	66.0 ± 1.1	10.88 ± 0.67	—	—	-3.4
H178F	0.44	3.38 ± 0.32	276.4 ± 6.4	81.83 ± 7.87	—	40.5 ± 0.1	-3.5
H178K	0.67	25.15 ± 1.69	22.5 ± 0.6	0.90 ± 0.06	—	—	8.9
H178N	2.59	7.36 ± 1.02	121.1 ± 4.9	16.46 ± 2.37	—	—	—
H178N	0.05	1.40 ± 0.51	273.1 ± 22.0	195.71 ± 72.72	—	—	—
H178N	0.85	5.64 ± 0.30	626.8 ± 9.8	111.21 ± 6.19	39.4 ± 0.2	—	3.3
H178Q	0.17	8.73 ± 0.50	231.4 ± 4.0	26.51 ± 1.59	—	—	2.6
H178R	—	—	—	—	—	—	—	²
H178R	0.09	4.96 ± 0.48	48.9 ± 1.4	9.85 ± 1.00	—	—	—
H178S	0.10	14.86 ± 0.61	51.0 ± 0.7	3.43 ± 0.15	—	—	—	³
H178W	—	—	—	—	—	—	—
H178W	0.72	10.31 ± 1.55	2.7 ± 0.1	0.27 ± 0.04	—	—	301.5
H178W	1.32	3.70 ± 0.58	660.6 ± 26.1	178.61 ± 28.91	—	—	20.2
H178Y	0.16	4.73 ± 0.24	4.0 ± 0.1	0.84 ± 0.04	—	—	-1.2
H182K	5.12	4.43 ± 0.35	377.2 ± 7.7	85.12 ± 6.87	—	50.0 ± 0.0	-0.8
H182Q	5.63	5.81 ± 0.38	412.3 ± 8.1	71.02 ± 4.90	—	49.5 ± 0.7	2.0
H182R	0.68	10.04 ± 0.98	720.0 ± 21.1	71.68 ± 7.28	—	—	—	³
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
H182S	1.18	4.26 ± 0.23	333.9 ± 8.3	78.41 ± 4.66	—	48.5 ± 0.7	2.7
H182T	4.02	3.72 ± 0.29	191.0 ± 9.4	51.36 ± 4.74	—	49.3 ± 1.2	8.1
H182V	—	—	—	—	—	—	6.6
H182Y	2.90	8.33 ± 2.15	528.0 ± 53.4	63.39 ± 17.61	—	47.2 ± 0.3	3.0
E183D	0.00	1.09 ± 0.12	885.3 ± 21.5	813.55 ± 90.21	39.6 ± 0.5	—	1.8
E183H	0.86	6.40 ± 0.55	355.9 ± 8.3	55.64 ± 4.93	—	—	—	³
E183T	4.10	4.50 ± 0.25	581.3 ± 8.6	129.17 ± 7.51	—	—	—	³
N184D	1.31	3.84 ± 0.44	1,109.4 ± 32.1	288.79 ± 33.88	40.4 ± 0.3	—	-0.2
E187A	8.07	5.94 ± 0.37	384.4 ± 8.6	64.76 ± 4.25	—	51.2 ± 0.6	2.3
E187F	6.13	5.32 ± 0.22	40.9 ± 0.5	7.69 ± 0.34	—	50.8 ± 0.6	1.8
E187T	2.84	6.15 ± 1.07	404.9 ± 20.1	65.81 ± 11.85	—	49.2 ± 0.8	5.4
A192S	*	5.09 ± 0.18	775.0 ± 8.7	152.19 ± 5.63	39.2 ± 0.3	—	—	²
H194G	0.20	1.59 ± 0.30	91.5 ± 4.4	57.56 ± 11.06	—	—	7.0
H194L	0.19	1.64 ± 0.29	136.1 ± 6.5	83.05 ± 15.34	—	—	28.9
H194L	0.29	3.91 ± 0.45	130.5 ± 3.8	33.40 ± 3.98	37.4 ± 0.2	—	—
H194N	0.85	4.31 ± 0.44	158.8 ± 4.2	36.86 ± 3.86	39.3 ± 0.5	—	—
H194N	1.29	4.71 ± 0.42	178.7 ± 4.2	37.98 ± 3.53	38.5 ± 0.4	—	—
H194Q	0.16	6.08 ± 0.86	78.8 ± 3.1	12.96 ± 1.90	37.2 ± 0.3	—	—
H194V	1.04	4.27 ± 0.53	194.4 ± 6.3	45.52 ± 5.88	39.9 ± 0.5	—	22.1
H194Y	0.73	4.85 ± 0.32	185.9 ± 3.3	38.32 ± 2.64	39.0 ± 0.4	—	—
H194Y	0.76	6.38 ± 1.08	187.6 ± 8.9	29.41 ± 5.17	38.8 ± 0.3	—	—
C198A	3.03	4.73 ± 0.47	82.9 ± 2.2	17.52 ± 1.79	39.3 ± 0.2	—	-0.9
C198D	—	—	—	—	—	—	6.8
C198F	—	—	—	—	—	—	287.9
C198G	0.93	1.59 ± 0.34	18.0 ± 0.8	11.33 ± 2.49	37.2 ± 0.4	—	4.1
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
C198K	1.76	4.96 ± 0.18	84.9 ± 0.8	17.13 ± 0.65	36.7 ± 1.3	—	55.6
C198L	0.68	—	—	—	—	—	33.2
C198M	1.24	4.93 ± 0.67	80.9 ± 2.9	16.42 ± 2.31	36.1 ± 0.4	—	42.9
C198N	—	—	—	—	—	—	12.0
C198Q	0.65	1.57 ± 0.32	2.8 ± 0.1	1.78 ± 0.38	40.9 ± 1.1	—	16.7
C198S	1.98	2.75 ± 0.39	113.1 ± 3.8	41.19 ± 5.96	38.6 ± 0.3	—	2.0
C198T	5.49	2.27 ± 0.24	132.1 ± 3.2	58.08 ± 6.26	35.9 ± 0.3	—	3.2
S203V	0.98	3.05 ± 0.46	1,281.5 ± 47.5	419.79 ± 65.64	44.3 ± 0.3	—	-4.1
T212E	1.14	4.99 ± 0.78	406.7 ± 17.0	81.56 ± 13.18	—	49.4 ± 0.2	-0.2
T212E	1.29	4.87 ± 0.40	361.5 ± 7.9	74.26 ± 6.34	—	43.5 ± 0.1	-0.2
I217S	1.08	5.46 ± 0.66	1,280.4 ± 42.3	234.72 ± 29.55	42.4 ± 0.1	—	6.4
T218A	*	6.51 ± 0.94	381.0 ± 15.2	58.45 ± 8.66	40.1 ± 0.1	47.5 ± 0.0	—	²
T218G	1.78	6.66 ± 1.34	142.0 ± 8.0	21.33 ± 4.45	—	43.0 ± 0.5	8.3
T218Q	1.04	—	—	0.06 ± 0.00	—	—	—
T218Q	4.40	5.49 ± 0.46	376.9 ± 8.6	68.61 ± 5.95	—	46.3 ± 0.7	22.8
T218S	1.11	14.39 ± 2.25	378.3 ± 20.2	26.28 ± 4.34	41.0 ± 0.2	—	—
T218S	3.00	30.64 ± 3.49	327.1 ± 15.9	10.68 ± 1.32	—	47.5 ± 0.3	—
L219M	0.82	5.58 ± 0.63	248.5 ± 7.6	44.54 ± 5.18	—	44.5 ± 0.3	—
L219Q	1.54	7.15 ± 0.16	1,266.0 ± 7.8	177.11 ± 4.03	—	—	—	³
L219Q	0.50	5.58 ± 3.50	173.1 ± 29.4	31.03 ± 20.15	—	—	6.6
L219R	3.02	2.79 ± 0.26	278.9 ± 6.2	100.01 ± 9.44	36.6 ± 0.3	—	—
L219V	1.38	3.46 ± 0.19	225.4 ± 3.1	65.05 ± 3.75	37.2 ± 0.1	—	—
N220A	*	10.27 ± 0.68	331.2 ± 6.5	32.26 ± 2.16	38.9 ± 0.2	43.6 ± 0.0	—	²
N220A	7.95	10.73 ± 1.23	83.8 ± 3.0	7.81 ± 0.94	38.7 ± 0.2	—	1.7
N220A	0.66	29.16 ± 17.68	71.3 ± 16.5	2.44 ± 1.59	—	—	1.7
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
N220A	0.59	11.24 ± 0.62	133.0 ± 2.3	11.84 ± 0.68	—	43.6 ± 0.3	1.7
N220C	0.78	5.87 ± 0.55	140.2 ± 3.9	23.89 ± 2.32	36.7 ± 0.4	—	—
N220C	1.05	4.82 ± 0.52	151.9 ± 4.3	31.54 ± 3.51	36.5 ± 0.1	—	—
N220C	1.39	1.76 ± 0.46	380.3 ± 21.9	215.89 ± 57.83	—	—	—
N220D	1.16	1.48 ± 0.30	100.3 ± 4.5	67.69 ± 14.11	35.0 ± 0.2	—	—
N220D	1.54	4.93 ± 0.37	9.0 ± 0.2	1.82 ± 0.14	35.6 ± 0.1	—	—
N220E	4.37	30.28 ± 3.18	32.8 ± 1.5	1.08 ± 0.12	35.8 ± 0.3	—	5.6
N220E	0.34	73.70 ± 13.07	371.5 ± 37.7	5.04 ± 1.03	36.7 ± 0.1	—	5.6
N220F	0.71	4.55 ± 0.61	607.9 ± 21.5	133.69 ± 18.61	39.2 ± 0.4	—	—
N220F	1.18	1.66 ± 0.16	94.6 ± 2.1	57.08 ± 5.58	36.1 ± 0.6	—	—
N220F	2.53	1.49 ± 0.07	134.6 ± 1.4	90.22 ± 4.34	37.6 ± 0.1	—	—
N220F	1.19	1.14 ± 0.09	81.5 ± 1.6	71.28 ± 5.90	—	—	—
N220F	2.62	0.52 ± 0.04	279.1 ± 4.0	540.60 ± 41.86	37.8 ± 34,740.5	—	—
N220F	2.12	0.52 ± 0.04	329.9 ± 4.6	636.56 ± 47.34	—	—	—
N220F	3.17	0.87 ± 0.05	1,532.3 ± 16.2	1,759.43 ± 93.88	—	—	—
N220F	2.12	0.52 ± 0.04	329.9 ± 4.6	636.56 ± 47.34	—	—	—
N220F	1.80	1.22 ± 0.14	678.7 ± 15.6	556.38 ± 64.67	38.7 ± 0.3	—	-2.0
N220F	—	—	—	—	—	—	-2.0
N220F	1.91	0.19 ± 0.02	224.2 ± 4.6	1,194.20 ± 141.17	38.1 ± 0.2	—	—
N220F	1.00	0.25 ± 0.02	84.6 ± 1.1	339.75 ± 24.73	—	—	—
N220G	*	33.30 ± 2.36	171.0 ± 4.3	5.20 ± 0.43	41.9 ± 0.2	47.4 ± 0.0	—	²
N220G	10.31	30.28 ± 3.18	13.9 ± 0.6	0.46 ± 0.05	35.8 ± 0.3	—	5.2
N220H	*	5.14 ± 0.21	101.7 ± 0.0	19.70 ± 0.87	39.6 ± 0.4	46.8 ± 0.0	—	²
N220H	7.46	4.48 ± 0.24	15.8 ± 0.2	3.52 ± 0.20	38.6 ± 0.2	—	0.0
N220I	*	—	—	—	36.7 ± 0.1	—	—
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
N220K	3.56	2.21 ± 0.41	351.6 ± 15.0	159.19 ± 30.49	—	—	—
N220K	2.67	—	—	—	40.0 ± 0.4	—	—
N220L	0.87	8.13 ± 1.49	641.1 ± 34.8	78.90 ± 15.10	39.2 ± 0.2	—	—
N220L	4.52	19.53 ± 4.90	8.2 ± 1.1	0.42 ± 0.12	34.2 ± 1.7	—	—
N220L	4.51	3.87 ± 0.27	456.5 ± 8.1	117.84 ± 8.47	38.2 ± 0.1	—	—
N220M	7.51	1.42 ± 0.09	100.7 ± 1.3	70.75 ± 4.41	37.2 ± 0.1	—	1.0
N220M	0.53	1.62 ± 0.15	352.5 ± 7.1	217.55 ± 20.40	35.3 ± 0.3	—	1.0
N220P	3.79	9.45 ± 0.46	77.1 ± 1.1	8.15 ± 0.41	35.6 ± 0.2	—	53.3
N220Q	0.29	4.46 ± 1.16	123.6 ± 9.0	27.72 ± 7.52	—	—	2.6
N220Q	6.39	6.11 ± 0.71	42.7 ± 1.4	6.99 ± 0.85	36.4 ± 0.2	—	—
N220R	*	12.50 ± 1.39	13.0 ± 0.0	1.08 ± 0.22	39.5 ± 0.3	46.9 ± 0.0	—	²
N220R	7.23	11.26 ± 1.96	2.9 ± 0.2	0.25 ± 0.05	41.6 ± 0.6	—	-14.2
N220R	0.59	6.33 ± 0.41	419.4 ± 7.7	66.29 ± 4.51	39.8 ± 0.3	—	-4.7
N220S	0.53	—	—	6.00 ± 0.19	39.7 ± 0.2	—	—
N220S	1.24	28.87 ± 2.65	7.3 ± 0.3	0.25 ± 0.03	41.0 ± 0.2	—	—
N220S	1.76	36.76 ± 1.55	130.5 ± 2.5	3.55 ± 0.16	—	47.2 ± 0.1	—
N220T	4.29	11.90 ± 1.23	0.4 ± 0.0	0.04 ± 0.00	40.0 ± 0.2	—	—
N220T	*	—	—	—	39.3 ± 0.3	—	—
N220V	1.30	2.98 ± 0.42	112.1 ± 3.8	37.68 ± 5.46	36.6 ± 0.3	—	54.0
N220V	1.30	2.98 ± 0.42	112.1 ± 3.8	37.68 ± 5.46	36.6 ± 0.3	—	54.0
N220W	0.58	0.52 ± 0.09	53.0 ± 2.0	102.74 ± 18.04	—	—	—	³
N220W	0.80	1.17 ± 0.30	277.7 ± 15.8	236.55 ± 62.52	—	—	—
N220Y	*	0.40 ± 0.02	515.2 ± 4.3	1,288.07 ± 65.38	41.7 ± 0.2	49.2 ± 0.0	—	²
N220Y	3.23	5.90 ± 0.20	447.9 ± 4.1	75.89 ± 2.60	—	—	—
N220Y	12.57	0.32 ± 0.04	85.9 ± 1.9	268.14 ± 34.07	39.9 ± 0.1	—	-2.3
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
M221A	*	6.25 ± 0.60	448.1 ± 13.0	71.66 ± 7.14	40.4 ± 0.4	47.2 ± 0.0	—	²
M221K	0.24	7.54 ± 0.58	101.7 ± 2.3	13.49 ± 1.08	—	46.5 ± 0.1	5.5
M221K	0.17	11.41 ± 0.60	126.8 ± 2.1	11.11 ± 0.61	—	46.4 ± 0.0	5.5
M221N	1.50	5.59 ± 0.33	246.3 ± 3.9	44.07 ± 2.67	36.6 ± 0.1	—	—
M221Q	2.84	8.03 ± 0.20	1,008.6 ± 7.3	125.56 ± 3.32	—	—	—	³
M221R	0.24	14.98 ± 0.66	25.1 ± 0.4	1.67 ± 0.08	—	—	3.2
E222A	*	0.63 ± 0.15	73.6 ± 4.3	116.90 ± 28.58	36.9 ± 0.1	36.7 ± 0.0	—	²
E222H	*	8.54 ± 0.53	132.1 ± 2.2	15.37 ± 1.08	34.9 ± 0.2	40.2 ± 0.0	—	²
E222K	*	7.22 ± 0.75	88.8 ± 2.2	12.34 ± 1.30	39.0 ± 0.4	—	—	²
E222Q	*	12.16 ± 0.65	547.7 ± 8.7	45.03 ± 2.60	39.8 ± 0.3	—	—	²
E222R	*	2.48 ± 0.44	34.6 ± 2.2	14.07 ± 2.60	39.1 ± 0.2	—	—	²
E222Y	*	18.43 ± 3.14	10.8 ± 0.0	0.65 ± 0.22	37.1 ± 0.5	—	—	²
H223A	2.52	4.75 ± 0.51	269.2 ± 7.6	56.63 ± 6.29	—	—	—
H223A	0.08	7.39 ± 2.12	778.0 ± 69.3	105.29 ± 31.63	—	—	—
H223A	0.49	3.16 ± 0.48	236.5 ± 9.3	74.75 ± 11.64	37.3 ± 1.2	41.6 ± 0.3	-2.5
H223A	4.09	8.02 ± 0.99	168.1 ± 6.3	20.96 ± 2.71	—	42.5 ± 0.2	—
H223S	4.60	6.49 ± 0.92	248.1 ± 9.9	38.21 ± 5.61	—	40.0 ± 0.1	1.2
P231K	2.98	4.57 ± 0.42	349.8 ± 8.5	76.53 ± 7.33	—	44.8 ± 0.2	0.2
D233I	0.58	3.94 ± 0.38	33.7 ± 1.1	8.55 ± 0.88	—	—	16.2
A236C	2.40	3.89 ± 0.44	202.5 ± 5.9	52.09 ± 6.13	—	39.4 ± 0.9	40.3
A236D	—	—	—	—	—	—	43.0
A236D	—	—	—	—	—	—	43.0
A236D	—	—	—	—	—	—	43.0
A236E	—	—	—	—	—	—	—	²
A236E	*	—	—	—	—	51.8 ± 0.4	160.3
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
A236S	0.13	—	—	0.01 ± 0.00	—	45.4 ± 0.8	6.7
A236V	1.55	0.52 ± 0.36	1.9 ± 0.2	3.62 ± 2.51	36.2 ± 1.3	—	—
A236V	0.50	0.38 ± 0.26	0.5 ± 0.1	1.39 ± 0.95	—	—	—
R239C	1.55	6.52 ± 0.41	161.3 ± 2.8	24.76 ± 1.61	—	—	6.7
R240A	1.10	14.50 ± 1.66	685.8 ± 26.9	47.30 ± 5.73	38.2 ± 0.2	—	2.3
R240A	3.30	10.93 ± 1.33	570.7 ± 22.1	52.23 ± 6.69	38.9 ± 0.1	—	2.3
R240A	3.30	10.14 ± 3.80	1.3 ± 0.1	8.28 ± 0.64	—	—	2.3
R240A	3.30	14.97 ± 1.19	450.9 ± 12.3	30.12 ± 2.53	—	44.8 ± 0.2	2.6
R240D	*	10.82 ± 0.47	231.6 ± 4.3	21.43 ± 1.08	37.2 ± 0.0	45.7 ± 0.0	—	²
R240E	—	—	—	—	—	—	—	²
R240G	0.30	23.66 ± 2.61	456.7 ± 19.1	19.31 ± 2.28	—	—	—
R240G	0.21	—	—	—	—	—	—
R240K	*	17.67 ± 3.32	736.0 ± 47.6	41.56 ± 8.23	39.3 ± 0.2	44.0 ± 0.0	—	²
R240K	2.20	8.17 ± 0.89	716.5 ± 23.2	87.70 ± 10.00	39.6 ± 0.2	—	2.2
D241H	0.28	9.97 ± 0.78	100.2 ± 2.4	10.05 ± 0.82	—	—	—	³
D241R	0.13	2.42 ± 0.46	5.1 ± 0.2	2.10 ± 0.41	—	—	1.3
F243H	0.72	37.31 ± 8.63	868.5 ± 91.5	23.28 ± 5.92	—	—	1.2
F243H	1.57	4.82 ± 0.46	236.4 ± 6.0	49.04 ± 4.88	39.4 ± 0.3	—	1.6
F243H	0.82	1.88 ± 0.50	558.7 ± 32.1	297.55 ± 81.46	—	—	2.8
F243H	1.24	0.61 ± 0.17	234.3 ± 12.8	382.57 ± 109.97	—	—	2.8
F243H	1.10	3.42 ± 0.71	598.4 ± 30.9	174.86 ± 37.48	—	—	2.8
F243H	*	—	—	—	42.2 ± 0.1	—	2.8
F243H	*	—	—	—	42.4 ± 0.2	—	2.8
F243H	*	—	—	—	42.0 ± 0.3	—	2.8
F243H	4.97	8.60 ± 2.81	351.8 ± 38.3	40.92 ± 14.09	—	46.0 ± 0.0	2.4
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
F243H	1.71	17.81 ± 1.54	546.2 ± 17.1	30.66 ± 2.81	—	45.9 ± 0.0	—
F243H	3.40	2.31 ± 0.61	427.9 ± 26.0	185.47 ± 50.21	—	—	-0.3
I244A	0.20	8.50 ± 1.59	451.3 ± 16.6	53.12 ± 10.12	40.4 ± 0.2	—	5.3
I244E	*	5.97 ± 1.04	407.0 ± 19.5	68.19 ± 12.34	37.8 ± 0.1	45.8 ± 0.0	—	²
I244K	3.48	14.07 ± 0.87	134.4 ± 2.8	9.55 ± 0.63	—	—	4.3
I244M	2.97	0.64 ± 0.35	179.0 ± 18.1	280.73 ± 156.82	—	—	—
I244M	2.97	4.80 ± 0.76	293.1 ± 12.4	61.06 ± 10.05	—	—	6.9
I244N	*	2.15 ± 0.13	223.0 ± 4.3	103.70 ± 6.49	36.8 ± 0.6	45.1 ± 0.0	—	²
I244Q	2.18	14.73 ± 0.94	1,166.6 ± 24.6	79.20 ± 5.31	—	—	—	³
I244T	3.11	4.22 ± 0.17	681.8 ± 6.9	161.68 ± 6.69	—	—	—	³
I244V	1.54	2.04 ± 0.16	518.2 ± 10.0	254.43 ± 21.17	—	—	—
R246K	0.47	0.45 ± 0.12	599.0 ± 30.9	1,340.16 ± 378.52	—	—	-0.6
R246K	0.29	0.50 ± 0.18	804.3 ± 57.5	1,624.32 ± 595.03	—	—	-0.6
R246K	0.48	0.53 ± 0.13	557.4 ± 25.3	1,060.53 ± 258.06	—	—	-0.6
R246K	0.56	2.46 ± 0.39	333.0 ± 11.4	135.56 ± 21.93	—	—	-0.6
R246K	0.41	3.13 ± 0.51	391.4 ± 15.5	125.24 ± 21.06	—	—	-0.6
R246K	0.42	1.56 ± 0.39	309.5 ± 17.0	198.59 ± 50.69	—	—	-0.6
R246K	*	—	—	—	36.8 ± 0.1	—	-0.6
R246K	*	—	—	—	36.7 ± 0.3	—	-0.6
R246K	*	—	—	—	37.3 ± 0.3	—	-0.6
R246K	*	—	—	—	36.6 ± 0.3	—	-0.6
R246K	*	—	—	—	37.0 ± 0.2	—	-0.6
R246K	*	—	—	—	36.6 ± 0.4	—	-0.6
R246K	2.75	6.43 ± 0.37	280.5 ± 4.5	43.64 ± 2.59	—	42.0 ± 0.4	-0.4
W247V	0.16	7.62 ± 2.81	4.2 ± 0.4	0.55 ± 0.21	—	—	—
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
F248N	0.30	8.99 ± 2.33	8.1 ± 0.6	0.91 ± 0.25	—	50.3 ± 0.7	6.2
A249E	—	—	—	—	—	—	—	²
E250D	0.77	1.21 ± 0.24	729.5 ± 30.9	604.21 ± 124.15	39.3 ± 0.7	—	-7.6
E250D	1.01	4.04 ± 0.48	903.3 ± 27.6	223.86 ± 27.64	40.8 ± 0.1	—	5.2
F253H	0.28	64.81 ± 42.05	297.9 ± 110.9	4.60 ± 3.44	—	—	3.6
F253I	1.25	24.22 ± 2.83	57.8 ± 2.7	2.38 ± 0.30	—	44.2 ± 0.2	6.6
F253I	4.06	5.68 ± 0.58	442.8 ± 12.3	78.01 ± 8.21	—	45.3 ± 0.0	5.2
F253I	2.36	6.79 ± 0.59	446.7 ± 11.1	65.78 ± 5.98	—	46.2 ± 0.4	—
F253M	1.00	—	—	—	—	—	6.8
F253M	3.35	5.99 ± 0.39	448.5 ± 8.2	74.93 ± 5.10	—	44.7 ± 0.1	6.9
F253Y	0.23	6.14 ± 0.61	271.2 ± 7.5	44.21 ± 4.56	—	—	1.4
F253Y	1.15	7.59 ± 0.51	279.9 ± 5.5	36.86 ± 2.58	—	40.9 ± 0.1	—
D260E	0.88	10.56 ± 0.84	1,349.4 ± 33.7	127.83 ± 10.64	—	—	—
M261D	—	—	—	—	—	—	—	²
W264H	0.27	10.37 ± 1.48	124.0 ± 6.0	11.96 ± 1.80	—	45.2 ± 0.0	0.0
W264Y	0.16	4.66 ± 2.54	24.4 ± 3.5	5.23 ± 2.96	—	—	—
Y265F	0.30	11.19 ± 3.84	5.5 ± 0.6	0.49 ± 0.18	34.5 ± 0.8	30.7 ± 18.7	-0.6
T267E	2.77	4.58 ± 0.47	422.5 ± 11.4	92.19 ± 9.79	—	47.6 ± 0.2	-1.3
Y268K	1.19	3.03 ± 0.36	1,237.6 ± 35.5	408.00 ± 49.49	39.7 ± 0.1	—	9.2
L269I	0.21	3.77 ± 0.53	176.9 ± 6.2	46.90 ± 6.80	40.4 ± 0.6	—	0.4
L269V	0.63	4.17 ± 0.73	898.7 ± 40.4	215.66 ± 38.85	40.6 ± 1.4	—	21.6
L269V	0.43	—	—	—	39.4 ± 0.6	—	21.6
N270G	1.31	11.70 ± 2.64	1,717.5 ± 135.9	146.81 ± 35.14	40.4 ± 0.1	—	3.2
N270G	0.98	2.60 ± 0.24	896.6 ± 19.6	345.48 ± 32.65	40.8 ± 0.2	—	3.2
N270W	0.60	1.02 ± 0.23	448.6 ± 20.7	441.69 ± 100.65	—	—	8.6
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
N270W	0.57	2.32 ± 0.38	344.4 ± 13.2	148.33 ± 25.02	—	—	8.6
N270W	0.44	4.20 ± 0.61	271.2 ± 10.1	64.60 ± 9.64	—	—	8.6
N270W	*	—	—	—	38.6 ± 0.1	—	8.6
N270W	*	—	—	—	39.7 ± 0.3	—	8.6
N270W	*	—	—	—	40.7 ± 0.5	—	8.6
N270W	2.85	5.65 ± 0.55	318.1 ± 8.5	56.27 ± 5.72	—	42.2 ± 0.1	8.5
G271D	0.25	2.47 ± 0.30	938.8 ± 28.9	379.65 ± 48.16	35.5 ± 0.2	—	4.7
G271D	0.61	1.27 ± 0.19	766.2 ± 23.9	601.75 ± 89.86	40.7 ± 0.3	—	4.7
G271D	0.53	0.48 ± 0.14	70.9 ± 3.6	147.45 ± 42.41	35.9 ± 0.2	—	—
L272F	1.23	0.80 ± 0.78	2.4 ± 0.5	3.01 ± 2.99	—	—	80.9
F274N	0.70	1.96 ± 0.22	156.5 ± 4.0	79.66 ± 9.10	34.9 ± 0.1	—	—
Q276K	0.74	3.48 ± 0.47	424.1 ± 14.2	121.94 ± 16.83	34.2 ± 0.9	—	4.6
G278V	3.54	5.16 ± 1.58	463.0 ± 36.2	89.76 ± 28.41	—	—	—
M280I	0.54	5.70 ± 0.43	451.7 ± 9.1	79.19 ± 6.22	—	—	—	³
D288E	2.29	5.38 ± 0.48	772.8 ± 18.0	143.61 ± 13.13	—	—	—	³
D288E	2.42	3.15 ± 0.38	267.7 ± 7.6	85.05 ± 10.47	—	44.0 ± 0.1	3.6
F289A	—	—	—	—	—	—	7.0
L290V	0.39	3.65 ± 0.41	1,461.3 ± 41.3	400.17 ± 46.29	—	44.3 ± 0.2	—
N293A	*	9.67 ± 0.44	10.8 ± 0.0	1.08 ± 0.00	40.8 ± 0.2	47.6 ± 0.0	—	²
N293A	0.79	13.78 ± 1.37	7.9 ± 0.3	0.57 ± 0.06	—	45.4 ± 0.1	-0.4
N293C	*	19.80 ± 2.08	8.7 ± 0.0	0.43 ± 0.00	38.9 ± 0.4	47.0 ± 0.0	—	²
N293D	—	—	—	—	—	—	—	²
N293E	*	—	—	—	—	—	18.9
N293F	—	—	—	—	—	—	86.3
N293G	6.13	23.71 ± 2.88	0.9 ± 0.0	0.04 ± 0.00	—	—	—	³
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
N293G	1.64	24.32 ± 2.71	1.3 ± 0.1	0.05 ± 0.01	—	—	—
N293H	*	—	—	—	—	42.1 ± 0.0	162.0
N293I	0.64	1.43 ± 0.28	1.3 ± 0.1	0.92 ± 0.19	39.6 ± 1.3	—	77.0
N293K	—	—	—	—	—	—	—	²
N293L	—	—	—	—	—	—	67.3
N293M	*	—	—	—	—	—	41.9
N293P	—	—	—	—	—	—	—
N293Q	*	—	—	—	—	—	—	²
N293R	0.37	0.70 ± 0.15	7.9 ± 0.3	11.25 ± 2.44	—	—	—	³
N293S	2.91	0.45 ± 0.18	2.2 ± 0.2	4.91 ± 1.96	—	47.8 ± 0.5	0.5
N293S	6.18	3.77 ± 0.62	3.4 ± 0.1	0.89 ± 0.15	41.3 ± 0.8	—	0.7
N293T	2.11	13.62 ± 1.65	29.2 ± 1.2	2.15 ± 0.27	38.3 ± 0.4	38.3 ± 0.4	11.8
N293V	2.05	8.72 ± 1.09	0.1 ± 0.0	0.01 ± 0.00	37.9 ± 67,349.9	—	—
N293V	1.24	4.87 ± 0.68	0.1 ± 0.0	0.01 ± 0.00	—	—	—
N293V	1.85	2.18 ± 0.85	7.9 ± 0.7	3.65 ± 1.45	—	43.4 ± 0.4	7.7
N293W	—	—	—	—	—	—	106.5
N293W	—	—	—	—	—	—	132.7
N293Y	0.61	48.85 ± 36.83	0.6 ± 0.2	0.01 ± 0.01	—	49.0 ± 0.8	—
N293Y	—	—	—	—	—	—	87.2
Y294A	*	4.98 ± 0.17	136.4 ± 2.2	27.49 ± 1.08	40.4 ± 0.2	43.3 ± 0.0	—	²
Y294F	*	5.99 ± 0.32	601.8 ± 8.7	100.45 ± 5.63	40.8 ± 0.1	44.0 ± 0.0	—	²
Y294H	*	—	—	—	—	—	3.2
Y294L	—	—	—	—	—	—	—	²
Y295A	*	—	—	—	—	—	—	²
Y295G	*	—	—	—	—	—	—	²
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
Y295N	1.59	18.93 ± 3.85	140.3 ± 10.5	7.41 ± 1.61	—	—	—
T296A	—	—	—	—	—	—	—	²
T296A	2.99	47.09 ± 4.63	0.6 ± 0.0	0.01 ± 0.00	—	—	—	³
T296C	1.03	12.12 ± 1.94	425.3 ± 21.5	35.10 ± 5.90	—	—	—	³
T296C	0.17	9.18 ± 0.80	71.3 ± 1.8	7.76 ± 0.70	—	—	—
T296E	—	—	—	—	—	—	—	²
T296F	5.25	0.84 ± 0.14	226.7 ± 7.7	269.30 ± 46.46	—	—	0.1
T296H	0.46	8.23 ± 1.10	101.5 ± 3.9	12.33 ± 1.72	—	—	—	³
T296R	0.91	1.27 ± 0.15	780.7 ± 23.1	615.34 ± 75.45	42.4 ± 0.1	—	0.8
T296S	2.46	3.91 ± 0.83	297.1 ± 18.2	76.00 ± 16.89	—	43.9 ± 0.3	0.4
T296W	2.62	0.66 ± 0.10	3.0 ± 0.1	4.49 ± 0.73	—	—	3.0
T296Y	4.35	5.95 ± 0.52	497.2 ± 12.0	83.60 ± 7.60	—	45.4 ± 0.0	-1.1
R297A	0.62	2.36 ± 0.49	52.2 ± 2.6	22.07 ± 4.74	41.8 ± 0.1	—	5.9
S298E	*	5.28 ± 0.05	662.4 ± 2.2	125.56 ± 1.30	39.6 ± 0.2	—	—	²
S298R	0.76	2.56 ± 1.14	435.2 ± 45.8	170.03 ± 77.66	39.4 ± 0.2	—	—
S298T	0.85	3.34 ± 0.37	766.1 ± 21.2	229.47 ± 26.45	39.4 ± 0.3	—	—
I299R	4.56	3.48 ± 0.55	263.8 ± 10.5	75.84 ± 12.43	—	—	—
I299R	4.56	3.48 ± 0.55	263.8 ± 10.5	75.84 ± 12.43	—	—	3.2
I300N	*	4.48 ± 0.32	569.3 ± 10.8	127.08 ± 9.31	38.6 ± 0.5	—	—	²
S302A	2.10	4.80 ± 0.28	306.6 ± 4.8	63.84 ± 3.83	—	44.7 ± 0.0	-0.8
S302A	2.16	4.85 ± 0.21	352.5 ± 4.0	72.72 ± 3.19	—	44.7 ± 0.0	—
S302D	6.77	14.89 ± 1.41	74.6 ± 2.4	5.01 ± 0.50	39.2 ± 0.2	—	5.9
S302F	6.64	5.01 ± 0.28	142.6 ± 2.1	28.48 ± 1.64	39.5 ± 0.1	—	6.2
S302F	9.03	2.58 ± 0.31	107.1 ± 3.1	41.52 ± 5.12	39.1 ± 0.1	—	6.2
S302H	5.23	6.24 ± 0.32	511.0 ± 7.2	81.83 ± 4.41	—	—	—	³
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
S302H	1.20	3.38 ± 0.41	625.7 ± 18.5	185.18 ± 23.20	—	—	—
S302K	6.91	5.72 ± 0.53	197.8 ± 5.0	34.56 ± 3.32	37.6 ± 0.5	—	9.8
S302N	3.27	5.43 ± 0.16	718.7 ± 5.4	132.24 ± 3.92	—	—	—	³
S302W	6.31	4.49 ± 1.15	248.4 ± 16.9	55.35 ± 14.66	38.9 ± 0.3	—	6.2
S302W	4.94	6.33 ± 0.46	137.8 ± 2.8	21.76 ± 1.62	38.8 ± 0.3	—	6.2
S302Y	11.07	5.53 ± 0.48	153.3 ± 3.7	27.74 ± 2.52	39.9 ± 0.1	—	5.8
T303K	1.17	3.45 ± 0.37	865.0 ± 23.3	250.97 ± 27.94	42.0 ± 0.1	—	76.0
A306K	1.55	4.71 ± 0.54	698.6 ± 21.3	148.21 ± 17.68	40.7 ± 0.2	—	-1.5
A306N	1.59	4.89 ± 0.18	301.5 ± 3.1	61.66 ± 2.39	—	44.6 ± 0.1	-2.1
A306N	1.77	5.04 ± 0.19	380.3 ± 3.8	75.43 ± 2.94	—	44.6 ± 0.0	—
A306Q	2.08	3.75 ± 0.63	587.8 ± 25.0	156.87 ± 27.23	37.6 ± 0.5	—	—
A306Q	2.08	3.75 ± 0.63	587.8 ± 25.0	156.87 ± 27.23	38.9 ± 1.0	—	—
A306S	0.63	11.26 ± 1.24	405.9 ± 14.3	36.06 ± 4.17	—	43.7 ± 0.1	-1.7
A306S	1.89	7.70 ± 0.68	514.4 ± 13.3	66.79 ± 6.15	40.4 ± 0.1	—	-1.3
A306S	1.85	7.04 ± 0.54	381.0 ± 8.3	54.09 ± 4.29	—	45.6 ± 0.4	—
A306S	0.48	1.72 ± 0.28	643.6 ± 24.1	373.43 ± 63.22	—	44.3 ± 0.0	-1.7
V311D	3.05	6.84 ± 0.65	295.0 ± 8.0	43.14 ± 4.25	—	41.8 ± 0.1	2.6
E312V	1.01	3.55 ± 0.15	834.1 ± 15.0	235.23 ± 10.68	—	—	3.7
E312V	0.87	2.73 ± 0.25	886.6 ± 19.6	325.18 ± 30.98	38.2 ± 0.3	—	4.7
E312V	0.72	3.84 ± 0.09	1,154.7 ± 9.1	300.89 ± 7.73	39.1 ± 0.1	—	2.6
Q313A	1.25	4.45 ± 0.57	603.3 ± 19.7	135.49 ± 17.81	39.6 ± 0.1	—	—
Q313C	1.60	4.72 ± 0.58	164.3 ± 5.3	34.81 ± 4.43	—	—	0.9
Q313C	4.68	6.37 ± 0.29	405.7 ± 5.2	63.69 ± 3.04	—	—	0.9
Q313E	4.20	6.76 ± 0.86	413.8 ± 14.9	61.19 ± 8.05	39.7 ± 0.1	—	—
Q313L	3.90	3.58 ± 0.23	363.5 ± 5.8	101.67 ± 6.76	40.1 ± 0.1	—	—
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
Q313N	0.37	6.14 ± 0.89	291.1 ± 11.4	47.42 ± 7.10	—	—	—	³
Q313P	2.63	3.48 ± 0.55	126.7 ± 4.7	36.42 ± 5.92	—	—	4.3
Q313R	*	3.58 ± 0.51	565.0 ± 19.5	157.82 ± 23.16	42.1 ± 0.3	—	—	²
Q313V	2.53	8.77 ± 0.68	900.2 ± 20.5	102.63 ± 8.35	—	—	—	³
V314A	2.85	2.64 ± 0.29	182.3 ± 4.8	69.19 ± 7.93	37.9 ± 0.4	—	2.8
V314D	8.32	5.14 ± 0.61	126.2 ± 4.1	24.58 ± 3.05	38.1 ± 0.2	—	6.7
V314E	6.38	2.42 ± 0.34	126.0 ± 4.4	52.01 ± 7.45	38.0 ± 0.1	—	2.8
V314F	5.00	4.47 ± 0.33	210.1 ± 4.0	46.98 ± 3.54	37.8 ± 0.2	—	8.2
V314H	6.95	3.43 ± 0.33	227.5 ± 5.8	66.32 ± 6.57	38.2 ± 0.1	—	5.6
V314K	2.94	5.55 ± 0.83	269.1 ± 12.3	48.45 ± 7.60	36.0 ± 0.4	—	2.4
V314L	7.15	0.97 ± 0.14	155.9 ± 4.5	161.20 ± 23.23	38.5 ± 0.2	—	2.3
V314L	8.89	2.67 ± 0.44	146.5 ± 5.8	54.84 ± 9.31	39.1 ± 0.3	—	2.2
V314M	7.63	6.76 ± 0.43	181.4 ± 3.3	26.84 ± 1.79	38.5 ± 0.2	—	4.3
V314Q	6.59	3.16 ± 0.49	165.6 ± 6.3	52.36 ± 8.31	38.5 ± 0.2	—	2.4
V314T	2.24	3.44 ± 0.37	164.7 ± 4.5	47.91 ± 5.38	38.5 ± 0.3	—	3.3
V314W	6.07	4.76 ± 0.20	168.1 ± 1.9	35.36 ± 1.58	37.1 ± 0.2	—	7.7
H315C	*	—	—	—	40.1 ± 0.2	—	—
H315C	0.37	2.98 ± 0.69	0.7 ± 0.0	0.23 ± 0.06	37.6 ± 1.0	—	—
H315D	0.32	0.68 ± 0.24	897.0 ± 54.3	1,312.84 ± 460.56	40.1 ± 0.1	—	—
H315E	*	—	—	—	—	—	—	²
H315E	1.78	2.84 ± 0.39	183.7 ± 6.8	64.70 ± 9.19	—	47.2 ± 0.1	-1.2
H315I	3.82	6.73 ± 0.32	588.1 ± 7.6	87.37 ± 4.25	—	—	—	³
H315K	0.38	6.14 ± 0.56	36.6 ± 0.9	5.96 ± 0.57	—	—	—	³
H315N	—	—	—	—	—	—	—	²
H315N	0.90	1.57 ± 0.26	531.6 ± 18.9	339.63 ± 58.36	40.4 ± 0.2	—	-1.5
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
H315N	1.42	1.60 ± 0.32	378.7 ± 15.8	236.82 ± 48.44	—	—	-1.5
H315N	1.05	1.97 ± 0.31	539.9 ± 20.5	273.92 ± 44.97	—	—	-1.5
H315N	1.02	0.79 ± 0.19	337.0 ± 16.0	427.62 ± 103.19	—	—	-1.5
H315N	0.77	1.39 ± 0.22	560.9 ± 18.9	402.15 ± 64.27	—	—	-1.5
H315N	0.66	1.18 ± 0.23	562.4 ± 23.5	478.02 ± 96.83	—	—	-1.5
H315N	0.62	0.92 ± 0.21	548.8 ± 26.0	595.13 ± 141.00	—	—	-1.5
H315N	*	—	—	—	40.4 ± 0.2	—	-1.5
H315N	*	—	—	—	40.7 ± 0.2	—	-1.5
H315N	*	—	—	—	40.5 ± 0.2	—	-1.5
H315N	*	—	—	—	41.2 ± 0.3	—	-1.5
H315N	*	—	—	—	41.3 ± 0.2	—	-1.5
H315N	*	—	—	—	41.0 ± 0.6	—	-1.5
H315P	0.84	5.39 ± 0.27	647.6 ± 9.8	120.14 ± 6.24	38.8 ± 0.2	—	-1.8
H315P	1.23	2.48 ± 0.47	219.2 ± 9.2	88.31 ± 17.02	—	43.1 ± 0.0	-1.3
H315Q	0.41	1.12 ± 0.78	6.6 ± 1.1	5.88 ± 4.26	40.4 ± 0.5	—	-1.1
H315T	4.76	4.67 ± 0.11	658.5 ± 4.0	141.04 ± 3.48	—	—	—	³
H315Y	0.90	7.02 ± 0.66	347.3 ± 9.4	49.47 ± 4.85	—	45.7 ± 0.1	—
M316K	0.35	3.77 ± 0.90	439.2 ± 25.9	116.43 ± 28.63	—	—	—	³
E318R	3.49	8.52 ± 0.97	5.3 ± 0.2	0.62 ± 0.07	—	—	—	³
T321S	1.84	9.44 ± 2.87	422.2 ± 42.1	44.72 ± 14.29	—	43.3 ± 0.4	2.4
M323A	*	9.34 ± 0.80	342.0 ± 8.7	36.59 ± 3.25	37.1 ± 0.1	42.5 ± 0.0	—	²
M323C	0.32	16.52 ± 1.78	105.5 ± 4.0	6.39 ± 0.73	39.9 ± 0.3	—	4.5
M323D	1.11	4.44 ± 0.89	5.4 ± 0.3	1.21 ± 0.25	—	40.3 ± 0.1	5.4
M323I	5.27	8.15 ± 0.35	344.0 ± 4.4	42.22 ± 1.90	—	44.9 ± 0.1	6.4
M323I	5.04	3.42 ± 0.31	309.9 ± 7.1	90.74 ± 8.58	36.1 ± 0.2	—	—
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
M323K	—	—	—	—	—	—	—	²
M323S	0.17	5.85 ± 0.43	476.8 ± 9.6	81.50 ± 6.18	38.6 ± 0.3	—	5.3
M323S	0.18	14.97 ± 1.83	529.1 ± 22.3	35.34 ± 4.58	38.6 ± 0.4	—	5.2
W325A	*	1.61 ± 0.23	23.8 ± 0.0	14.72 ± 2.16	40.1 ± 0.5	41.8 ± 0.0	—	²
W325A	0.31	0.23 ± 0.16	80.6 ± 9.5	355.47 ± 253.21	32.7 ± 2,998.2	32.0 ± 1.1	7.0
W325C	*	4.18 ± 0.53	8.7 ± 0.0	2.16 ± 0.22	39.3 ± 0.2	41.1 ± 0.0	—	²
W325D	2.22	27.09 ± 3.65	1,039.5 ± 55.4	38.38 ± 5.57	—	—	—	³
W325E	3.58	2.26 ± 0.28	1.6 ± 0.1	0.71 ± 0.09	38.0 ± 0.3	—	5.2
W325F	1.87	12.63 ± 3.27	116.0 ± 9.7	9.19 ± 2.50	—	—	—	³
W325G	—	—	—	—	—	—	—	²
W325G	*	—	—	—	39.0 ± 0.6	—	10.0
W325H	*	3.08 ± 0.43	28.1 ± 0.0	9.09 ± 1.30	37.7 ± 0.3	40.7 ± 0.0	—	²
W325I	3.45	1.29 ± 0.16	6.2 ± 0.2	4.84 ± 0.61	39.6 ± 0.5	—	29.7
W325K	0.48	2.14 ± 0.67	1.0 ± 0.1	0.49 ± 0.16	—	—	—
W325K	0.90	9.24 ± 1.62	15.2 ± 0.8	1.64 ± 0.30	—	—	—
W325K	0.48	2.14 ± 0.67	1.0 ± 0.1	0.49 ± 0.16	—	—	—
W325K	0.30	0.83 ± 0.50	0.9 ± 0.1	1.04 ± 0.64	—	—	3.6
W325K	1.54	2.57 ± 0.23	11.3 ± 0.3	4.40 ± 0.42	39.4 ± 0.4	—	—
W325L	*	5.74 ± 0.35	88.8 ± 2.2	15.37 ± 1.08	40.4 ± 0.5	—	—	²
W325M	1.67	4.88 ± 0.75	43.3 ± 1.7	8.86 ± 1.40	—	43.4 ± 0.5	3.5
W325M	4.40	6.77 ± 0.28	60.6 ± 0.7	8.96 ± 0.38	—	42.3 ± 0.1	3.5
W325M	7.66	4.43 ± 0.36	21.3 ± 0.5	4.81 ± 0.41	38.8 ± 0.1	—	—
W325M	1.47	6.48 ± 4.56	31.1 ± 5.8	4.80 ± 3.50	—	45.8 ± 0.2	3.5
W325N	3.86	1.93 ± 0.15	4.4 ± 0.1	2.27 ± 0.18	40.4 ± 0.4	—	6.2
W325P	—	—	—	—	—	—	112.0
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
W325Q	1.43	1.02 ± 0.23	3.3 ± 0.2	3.22 ± 0.76	39.1 ± 0.3	—	3.9
W325Q	2.61	0.95 ± 0.19	14.7 ± 0.6	15.40 ± 3.07	—	42.2 ± 0.0	3.9
W325Q	0.30	0.33 ± 0.09	67.3 ± 3.2	203.06 ± 55.33	—	—	3.9
W325Q	0.25	0.32 ± 0.11	74.8 ± 4.4	232.77 ± 77.80	—	—	3.9
W325Q	0.36	0.38 ± 0.11	59.4 ± 3.2	155.22 ± 46.65	—	—	3.9
W325Q	0.37	—	—	—	—	—	3.9
W325Q	*	—	—	—	39.9 ± 0.2	—	3.9
W325Q	*	—	—	—	40.1 ± 0.2	—	3.9
W325R	*	—	—	—	40.0 ± 0.2	42.7 ± 0.0	—	²
W325R	2.05	14.18 ± 3.12	213.4 ± 15.7	15.05 ± 3.49	—	—	—
W325R	—	—	—	—	—	—	3.0
W325S	1.89	1.05 ± 0.21	6.1 ± 0.2	5.81 ± 1.17	42.0 ± 1.5	—	7.2
W325T	3.96	2.03 ± 0.26	7.0 ± 0.2	3.45 ± 0.46	38.2 ± 0.5	—	9.0
W325V	3.50	1.42 ± 0.16	5.6 ± 0.1	3.92 ± 0.45	39.1 ± 0.7	—	—
W325V	1.68	1.43 ± 0.15	11.7 ± 0.3	8.17 ± 0.91	39.1 ± 0.7	—	41.5
W325Y	3.02	2.41 ± 0.20	32.6 ± 0.6	13.52 ± 1.16	40.0 ± 0.2	—	5.1
W325Y	*	—	—	—	41.5 ± 0.3	—	—
H328A	0.77	8.24 ± 0.32	423.2 ± 4.9	51.36 ± 2.09	—	45.4 ± 0.1	—
H328K	1.70	13.77 ± 4.10	560.8 ± 59.9	40.72 ± 12.89	39.2 ± 0.3	—	—
H328K	1.60	0.82 ± 0.26	116.9 ± 9.0	141.81 ± 45.47	38.5 ± 0.4	—	1.1
H328K	2.20	3.94 ± 0.36	242.4 ± 5.6	61.47 ± 5.73	39.2 ± 0.3	—	—
H328P	0.36	2.71 ± 0.67	4.3 ± 0.2	1.58 ± 0.40	—	—	51.0
P329N	—	—	—	—	—	—	—	²
S331A	*	4.34 ± 0.11	668.9 ± 4.3	154.14 ± 4.11	39.3 ± 0.3	41.0 ± 0.0	—	²
S331G	2.44	5.82 ± 0.16	1,425.9 ± 14.4	244.86 ± 7.02	—	—	—	³
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
S331T	0.89	4.50 ± 1.38	27.4 ± 1.9	6.09 ± 1.91	—	—	—
Y333F	2.65	2.01 ± 0.19	185.7 ± 4.1	92.18 ± 9.15	37.8 ± 59,417.3	—	—
Y333F	1.55	1.75 ± 0.21	685.3 ± 18.2	392.17 ± 47.69	37.8 ± 60,350.4	—	—
Y333F	2.65	2.01 ± 0.19	185.7 ± 4.1	92.18 ± 9.15	—	—	—
Y333F	2.74	1.77 ± 0.47	320.0 ± 17.7	181.03 ± 49.22	—	43.4 ± 0.3	0.3
Y333F	3.85	7.29 ± 0.59	314.6 ± 7.3	43.17 ± 3.61	—	43.3 ± 0.3	0.3
K334D	1.20	6.11 ± 0.90	279.9 ± 11.1	45.78 ± 6.95	—	—	—	³
L336S	0.28	8.02 ± 0.47	189.2 ± 3.1	23.58 ± 1.43	—	—	—	³
E340A	1.00	2.64 ± 0.24	608.7 ± 13.1	230.43 ± 21.38	38.2 ± 0.2	—	4.0
E340A	2.70	0.53 ± 0.20	208.4 ± 16.4	393.41 ± 154.33	38.4 ± 0.2	—	-17.6
E340A	3.42	2.96 ± 0.39	268.4 ± 9.3	90.57 ± 12.39	—	45.1 ± 0.1	4.0
E340S	0.81	3.54 ± 0.46	308.7 ± 10.1	87.21 ± 11.72	—	45.0 ± 0.3	4.4
K341A	*	5.46 ± 0.33	857.3 ± 13.0	156.95 ± 9.74	40.0 ± 0.1	—	—	²
K341A	3.14	5.20 ± 0.40	363.8 ± 7.6	70.02 ± 5.64	—	45.1 ± 0.5	2.9
F343E	—	—	—	—	—	—	-3.2
F343E	0.10	45.41 ± 19.90	29.0 ± 6.2	0.64 ± 0.31	—	—	-3.1
F343S	—	—	—	—	—	—	—	²
L350I	1.67	5.71 ± 0.48	535.9 ± 11.9	93.83 ± 8.09	—	—	—	³
L350I	3.41	3.71 ± 0.39	27.4 ± 0.8	7.38 ± 0.80	—	47.7 ± 0.1	0.1
I351V	3.95	4.78 ± 0.39	436.1 ± 9.2	91.20 ± 7.74	—	—	—	³
T352A	*	14.26 ± 1.76	49.8 ± 2.2	3.46 ± 0.43	38.1 ± 0.3	39.3 ± 0.0	—	²
T352A	0.03	—	—	—	—	—	1.9
T352A	0.90	8.74 ± 0.51	116.4 ± 2.0	13.31 ± 0.81	—	41.0 ± 0.4	—
T352G	0.43	6.83 ± 0.37	21.1 ± 0.4	3.09 ± 0.18	—	—	4.4
T352P	0.24	5.21 ± 0.36	252.6 ± 4.6	48.50 ± 3.42	—	—	—
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
T352S	0.23	5.11 ± 1.38	234.4 ± 18.2	45.83 ± 12.86	37.5 ± 0.3	—	—
T352S	—	—	—	—	—	—	—
T352V	0.05	2.68 ± 0.45	79.1 ± 3.1	29.52 ± 5.06	—	—	—
T352V	0.05	2.68 ± 0.45	79.1 ± 3.1	29.52 ± 5.06	—	—	—
T352V	0.07	—	—	—	—	—	—
T352W	—	—	—	—	—	—	71.5
E353A	*	—	—	—	—	—	—	²
E353D	0.45	2.35 ± 0.99	1.6 ± 0.2	0.68 ± 0.29	—	—	—
N354A	*	5.38 ± 0.67	2.2 ± 0.0	0.43 ± 0.00	40.8 ± 0.4	46.7 ± 0.0	—	²
N354S	0.25	2.12 ± 0.39	67.4 ± 2.8	31.70 ± 5.98	—	—	1.8
N354S	1.01	4.55 ± 0.49	41.9 ± 1.2	9.21 ± 1.03	—	40.9 ± 0.3	—
G355A	—	—	—	—	—	—	—	²
G355N	0.32	8.65 ± 2.96	0.4 ± 0.0	0.04 ± 0.01	—	—	-3.6
A356T	1.90	2.89 ± 0.18	285.4 ± 4.2	98.63 ± 6.22	—	45.1 ± 0.1	—
M358T	*	4.83 ± 0.48	357.2 ± 8.7	74.04 ± 7.58	39.5 ± 0.2	—	—	²
L362M	*	5.20 ± 0.19	751.2 ± 6.5	144.39 ± 5.41	40.0 ± 0.2	—	—	²
V363N	2.07	4.64 ± 0.32	478.2 ± 8.3	103.16 ± 7.24	—	—	—	³
V363Q	2.47	5.91 ± 0.26	57.2 ± 0.7	9.68 ± 0.44	—	42.5 ± 0.5	1.3
V363Q	1.14	5.44 ± 0.52	348.1 ± 9.1	64.04 ± 6.37	—	41.4 ± 0.1	1.9
G365N	3.39	4.77 ± 0.12	961.6 ± 8.5	201.64 ± 5.33	—	—	—	³
R372F	0.41	4.84 ± 1.70	1.0 ± 0.1	0.21 ± 0.08	—	—	5.8
R372H	3.57	3.73 ± 0.33	625.2 ± 14.0	167.65 ± 15.29	—	—	2.9
R372W	0.51	0.56 ± 0.22	0.3 ± 0.0	0.56 ± 0.22	—	—	9.0
R372Y	0.42	2.88 ± 0.47	1.7 ± 0.1	0.58 ± 0.10	—	—	6.8
H373R	*	6.31 ± 0.30	580.2 ± 6.5	92.00 ± 4.55	39.2 ± 0.3	51.4 ± 0.0	—	²
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
Y375H	0.49	5.45 ± 0.36	418.7 ± 7.4	76.84 ± 5.30	—	—	—	³
Y375T	0.43	—	—	0.09 ± 0.00	—	—	8.2
E377Q	0.31	4.86 ± 0.69	1,537.4 ± 58.0	316.60 ± 46.56	39.6 ± 0.2	—	—
E377S	1.11	4.64 ± 0.69	590.6 ± 22.4	127.35 ± 19.44	—	—	—	³
H379R	*	—	—	—	—	—	—	²
H379T	—	—	—	—	—	—	—	²
H379Y	1.05	5.02 ± 0.49	35.3 ± 0.9	7.03 ± 0.70	—	—	-1.1
H379Y	0.57	0.22 ± 0.08	343.7 ± 22.7	1,549.66 ± 583.37	39.3 ± 0.3	—	-0.9
H379Y	0.75	2.43 ± 0.96	274.0 ± 18.6	112.61 ± 44.89	—	—	-0.9
H379Y	0.89	6.10 ± 1.80	313.4 ± 23.3	51.40 ± 15.64	—	—	-0.9
H379Y	0.42	0.30 ± 0.14	371.4 ± 27.3	1,229.79 ± 568.82	—	—	-0.9
H379Y	0.45	0.75 ± 0.46	72.1 ± 5.6	95.69 ± 58.61	—	—	-0.9
H379Y	0.48	5.28 ± 1.80	98.6 ± 8.2	18.68 ± 6.56	—	—	-0.9
H379Y	0.26	0.05 ± 0.10	97.8 ± 24.6	1,962.96 ± 3,948.07	—	—	-0.9
H379Y	*	—	—	—	—	—	-0.9
H379Y	*	—	—	—	—	—	-0.9
H379Y	*	—	—	—	—	—	-0.9
H379Y	*	—	—	—	—	—	-0.9
H379Y	*	—	—	—	—	—	-0.9
H384A	1.09	6.22 ± 0.42	644.9 ± 11.9	103.69 ± 7.30	—	—	—	³
H384I	2.95	2.23 ± 0.26	176.1 ± 5.1	79.13 ± 9.56	—	40.1 ± 0.1	12.9
H384I	3.89	3.24 ± 0.34	190.3 ± 5.3	58.76 ± 6.46	—	40.2 ± 0.2	12.9
H384R	0.43	6.00 ± 0.39	31.4 ± 0.5	5.24 ± 0.35	—	—	—	³
I387K	0.17	9.29 ± 1.08	94.5 ± 3.4	10.18 ± 1.24	—	43.6 ± 0.4	4.7
E388F	3.62	4.79 ± 0.37	317.7 ± 6.5	66.28 ± 5.31	—	—	—
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
E388G	5.11	1.62 ± 0.21	177.9 ± 5.0	109.58 ± 14.28	—	44.6 ± 0.1	-4.4
E388H	0.76	—	—	187.41 ± 4.88	39.7 ± 0.1	—	-3.3
E388H	0.82	3.09 ± 0.80	382.2 ± 24.3	123.53 ± 33.02	41.0 ± 0.2	—	-3.3
E388H	0.74	4.79 ± 0.26	108.7 ± 1.8	22.70 ± 1.30	39.7 ± 0.2	—	-3.3
E388L	3.13	6.62 ± 1.23	71.6 ± 3.8	10.81 ± 2.09	—	—	—
E388L	3.79	3.95 ± 0.30	201.5 ± 3.9	50.99 ± 3.97	40.2 ± 0.2	—	—
E388L	2.19	0.33 ± 0.11	66.7 ± 4.5	199.25 ± 67.22	—	44.9 ± 0.4	-0.4
E388R	4.14	3.67 ± 0.80	304.6 ± 16.4	82.96 ± 18.62	—	—	—
E389R	*	—	—	—	—	45.2 ± 0.1	-0.7
Q392N	0.47	4.84 ± 1.10	331.4 ± 21.1	68.41 ± 16.07	—	—	—
Y396C	—	—	—	—	—	—	0.9
Y396D	—	—	—	—	—	—	9.4
Y396E	—	—	—	—	—	—	11.3
Y396F	1.14	6.40 ± 1.03	155.0 ± 7.0	24.23 ± 4.03	38.0 ± 0.2	—	1.8
Y396G	—	—	—	—	—	—	14.6
Y396P	—	—	—	—	—	—	283.8
Y396P	—	—	—	—	—	—	283.8
Y396Q	—	—	—	—	—	—	9.0
Y396R	—	—	—	—	—	—	9.3
Y396S	—	—	—	—	—	—	8.2
Y396V	—	—	—	—	—	—	21.5
Y396W	1.28	1.96 ± 0.54	14.1 ± 1.1	7.20 ± 2.05	38.4 ± 0.5	—	3.9
F397A	0.68	2.78 ± 0.61	25.1 ± 1.4	9.03 ± 2.03	39.2 ± 4.4	39.1 ± 2.9	6.7
F397D	—	—	—	—	—	—	7.9
F397E	—	—	—	—	—	—	0.0
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
F397G	—	—	—	—	—	—	12.0
F397H	—	—	—	—	—	—	3.8
F397I	2.20	2.02 ± 0.28	79.4 ± 2.5	39.24 ± 5.56	37.5 ± 0.1	37.2 ± 0.8	26.9
F397K	—	—	—	—	—	—	0.0
F397N	0.71	8.68 ± 0.95	86.3 ± 3.1	9.94 ± 1.14	38.5 ± 0.8	38.5 ± 0.8	6.0
F397R	—	—	—	—	—	—	8.6
F397S	1.10	4.81 ± 0.23	88.0 ± 1.1	18.31 ± 0.91	35.7 ± 0.7	35.7 ± 0.7	7.6
F397V	0.50	3.01 ± 0.44	7.1 ± 0.2	2.38 ± 0.36	36.7 ± 0.3	—	27.7
F397W	1.37	9.02 ± 0.79	105.0 ± 3.3	11.64 ± 1.09	38.2 ± 0.4	38.2 ± 0.4	0.5
F397Y	—	—	—	—	—	—	1.6
V398N	0.41	4.11 ± 0.25	400.5 ± 6.3	97.37 ± 6.16	34.3 ± 0.2	—	4.6
W399A	*	—	—	—	—	—	—	²
W399C	*	70.33 ± 5.89	2.2 ± 0.0	—	41.7 ± 0.1	44.8 ± 0.0	—	²
W399G	*	—	—	—	—	—	—	²
W399Q	3.85	21.84 ± 3.20	0.6 ± 0.0	0.03 ± 0.00	—	44.8 ± 0.1	6.4
W399R	*	12.75 ± 0.87	—	—	37.9 ± 0.8	49.1 ± 0.0	—	²
W399S	*	—	—	—	—	—	—	²
S400A	*	3.22 ± 0.22	435.1 ± 6.5	135.09 ± 9.53	39.9 ± 0.3	47.3 ± 0.0	—	²
S400G	2.19	30.88 ± 2.17	287.8 ± 8.3	9.32 ± 0.71	—	—	—	³
S400W	—	—	—	—	—	—	124.8
F401K	0.63	—	—	—	—	44.9 ± 0.8	18.4
L402C	5.89	3.12 ± 0.21	744.3 ± 24.0	238.68 ± 17.68	—	—	—	³
L402E	0.27	2.58 ± 0.14	43.7 ± 0.6	16.96 ± 0.95	—	—	—	³
D403A	—	—	—	—	—	—	—	²
N404A	*	9.42 ± 0.42	4.3 ± 0.0	0.43 ± 0.00	41.9 ± 0.1	54.4 ± 0.0	—	²
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
N404A	3.83	6.09 ± 1.77	1.2 ± 0.1	0.20 ± 0.06	—	—	—
N404C	*	14.40 ± 1.90	69.3 ± 2.2	4.76 ± 0.65	42.7 ± 0.2	46.0 ± 0.0	—	²
N404C	1.55	23.13 ± 4.28	86.6 ± 6.0	3.75 ± 0.74	43.7 ± 0.2	—	-1.9
N404D	1.19	49.80 ± 3.48	18.7 ± 0.7	0.38 ± 0.03	—	—	—
N404D	1.69	4.43 ± 0.65	550.8 ± 21.1	124.39 ± 18.90	—	42.4 ± 1.8	5.7
N404E	1.76	8.76 ± 1.25	0.8 ± 0.0	0.09 ± 0.01	—	45.2 ± 1.0	—
N404E	1.96	2.57 ± 1.09	0.3 ± 0.0	0.10 ± 0.04	41.0 ± 4.2	—	4.7
N404E	0.22	7.16 ± 1.11	3.8 ± 0.2	0.53 ± 0.09	—	—	—
N404E	4.32	3.52 ± 0.40	250.8 ± 7.1	71.21 ± 8.29	—	46.1 ± 0.2	3.9
N404E	0.47	3.19 ± 0.49	11.6 ± 0.4	3.62 ± 0.57	40.0 ± 0.4	44.7 ± 0.1	3.9
N404L	1.99	31.24 ± 2.14	15.7 ± 0.5	0.50 ± 0.04	—	42.9 ± 0.0	-1.5
N404L	2.78	27.89 ± 2.63	21.2 ± 0.8	0.76 ± 0.08	—	46.0 ± 0.2	-1.7
N404L	0.94	33.10 ± 1.23	31.5 ± 0.5	0.95 ± 0.04	—	44.9 ± 0.1	-1.5
N404M	2.39	10.39 ± 2.30	53.1 ± 3.7	5.11 ± 1.18	—	—	-3.4
N404M	1.87	57.08 ± 3.60	7.3 ± 0.2	0.13 ± 0.01	—	—	-3.4
N404M	0.58	14.11 ± 1.07	7.2 ± 0.2	0.51 ± 0.04	—	47.8 ± 0.3	-3.0
N404M	2.02	15.24 ± 2.74	7.4 ± 0.5	0.49 ± 0.09	—	48.9 ± 0.1	-3.0
N404M	4.00	10.66 ± 0.99	15.5 ± 0.5	1.45 ± 0.14	40.6 ± 0.3	—	-3.4
N404M	2.50	14.36 ± 0.96	7.4 ± 0.2	0.51 ± 0.04	—	44.9 ± 0.1	—
N404M	2.00	13.72 ± 2.68	10.9 ± 0.7	0.80 ± 0.16	—	45.8 ± 0.0	-3.0
N404M	1.23	—	—	—	—	45.6 ± 0.1	—
N404Q	2.43	1.43 ± 0.58	0.1 ± 0.0	0.08 ± 0.04	—	48.1 ± 0.1	—
N404Q	3.71	5.31 ± 1.19	0.1 ± 0.0	0.02 ± 0.01	—	46.0 ± 0.5	0.4
N404Q	0.57	0.70 ± 0.46	0.2 ± 0.0	0.31 ± 0.21	—	50.8 ± 0.1	0.6
N404S	4.96	23.55 ± 5.86	23.4 ± 2.2	0.99 ± 0.26	—	—	—	³
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
N404S	0.76	30.04 ± 1.50	47.0 ± 1.0	1.56 ± 0.08	44.5 ± 0.1	—	—
E406A	*	—	—	—	40.1 ± 0.7	49.1 ± 0.0	—	²
E406D	*	34.13 ± 2.57	32.5 ± 0.0	0.87 ± 0.00	40.7 ± 0.3	42.2 ± 0.0	—	²
E406D	0.49	24.18 ± 5.30	24.4 ± 2.1	1.01 ± 0.24	40.2 ± 0.2	—	-4.4
E406F	0.64	—	—	—	—	—	—
E406H	2.36	0.02 ± 0.04	0.0 ± 0.0	1.80 ± 2.83	—	—	4.5
E406K	*	—	—	—	—	44.1 ± 0.3	25.2
E406Q	1.50	69.03 ± 5.81	8.8 ± 0.4	0.13 ± 0.01	—	44.4 ± 0.6	4.3
E406Q	4.46	38.26 ± 1.65	5.9 ± 0.1	0.15 ± 0.01	—	44.1 ± 0.2	4.3
E406Q	4.70	67.24 ± 6.50	96.0 ± 5.1	1.43 ± 0.16	—	53.2 ± 2.9	—
E406Q	4.47	—	—	0.07 ± 0.00	—	51.5 ± 0.0	—
E406Q	3.90	—	—	0.05 ± 0.00	—	42.8 ± 0.2	—
E406T	3.40	2.57 ± 1.38	15.6 ± 1.9	6.05 ± 3.33	—	—	—	³
W407A	*	—	—	—	—	—	—	²
W407A	0.91	27.09 ± 4.32	0.6 ± 0.0	0.02 ± 0.00	—	42.4 ± 0.2	—
W407D	0.14	0.37 ± 0.90	3.0 ± 1.4	8.05 ± 20.19	—	—	2.5
W407D	0.81	7.09 ± 4.72	0.2 ± 0.0	0.02 ± 0.02	—	42.4 ± 0.2	—
W407D	0.76	—	—	0.00 ± 0.00	—	44.3 ± 0.9	-3.2
W407E	—	—	—	—	—	—	10.6
W407F	2.54	32.02 ± 3.82	4.1 ± 0.2	0.13 ± 0.02	39.2 ± 0.6	—	—
W407F	0.98	38.81 ± 4.02	6.1 ± 0.3	0.16 ± 0.02	—	44.9 ± 0.0	6.1
W407F	1.88	30.45 ± 1.93	7.9 ± 0.2	0.26 ± 0.02	—	42.8 ± 0.1	4.1
W407G	—	—	—	—	—	—	—	²
W407H	1.45	0.03 ± 0.02	0.1 ± 0.0	1.96 ± 1.12	—	—	—
W407H	0.50	11.12 ± 4.31	0.9 ± 0.1	0.08 ± 0.03	—	—	—
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
W407K	—	—	—	—	—	—	—	²
W407K	0.09	17.69 ± 3.06	3.6 ± 0.2	0.20 ± 0.04	—	—	—	³
W407K	—	—	—	—	—	—	3.5
W407L	*	—	—	—	—	—	29.5
W407N	0.25	—	—	—	—	—	-0.5
W407N	0.15	—	—	—	—	—	-1.4
W407Q	—	—	—	—	—	—	—	²
W407R	—	—	—	—	—	—	—	²
W407R	—	—	—	—	—	—	8.8
W407S	—	—	—	—	—	—	-1.8
W407Y	—	—	—	—	—	—	—	²
A408F	0.31	37.97 ± 8.36	341.9 ± 33.2	9.00 ± 2.17	—	—	—	³
A408H	0.36	—	—	—	—	—	6.0
A408H	1.50	12.85 ± 1.04	246.9 ± 6.6	19.21 ± 1.63	—	43.6 ± 0.0	0.0
A408H	1.61	13.76 ± 0.67	270.9 ± 4.5	19.69 ± 1.01	—	43.8 ± 0.1	—
A408K	2.59	6.32 ± 0.92	319.8 ± 12.7	50.56 ± 7.62	—	—	—	³
A408K	2.88	4.02 ± 0.37	312.1 ± 7.3	77.68 ± 7.34	—	48.2 ± 0.3	—
A408K	0.39	5.69 ± 0.61	802.9 ± 23.6	141.06 ± 15.68	—	44.6 ± 0.1	—
A408S	0.23	9.32 ± 2.00	158.5 ± 10.3	17.02 ± 3.81	38.4 ± 0.2	—	1.9
A408T	0.14	12.90 ± 2.77	153.9 ± 10.7	11.93 ± 2.69	37.3 ± 0.5	—	4.9
W409A	4.66	4.69 ± 0.72	117.3 ± 4.6	25.02 ± 3.97	39.8 ± 0.3	—	3.6
W409A	0.45	6.33 ± 1.14	170.3 ± 8.2	26.92 ± 5.02	40.0 ± 0.2	—	4.2
W409A	0.67	10.42 ± 2.02	264.5 ± 16.1	25.38 ± 5.16	40.7 ± 0.3	—	4.2
W409C	6.00	2.44 ± 0.13	156.2 ± 1.9	64.01 ± 3.39	39.4 ± 2.2	—	5.0
W409D	3.60	2.29 ± 0.14	532.9 ± 7.4	233.16 ± 14.49	—	—	—	³
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
W409D	0.24	73.27 ± 12.71	527.8 ± 52.2	7.20 ± 1.44	40.1 ± 0.3	—	6.8
W409D	0.61	2.33 ± 0.21	715.4 ± 14.8	307.39 ± 27.92	40.6 ± 0.2	—	6.8
W409E	0.78	2.12 ± 0.10	686.6 ± 9.2	323.18 ± 16.03	—	—	—
W409E	0.78	2.21 ± 0.09	910.6 ± 10.6	412.37 ± 17.64	—	—	—
W409E	2.59	1.99 ± 0.24	312.5 ± 8.6	157.26 ± 19.52	—	46.8 ± 0.1	4.9
W409E	2.35	0.91 ± 0.09	102.8 ± 2.1	112.40 ± 11.23	39.6 ± 0.4	—	4.9
W409E	2.10	2.99 ± 0.36	299.6 ± 8.7	100.12 ± 12.28	—	42.6 ± 0.1	—
W409F	0.78	3.65 ± 0.29	615.2 ± 12.3	168.53 ± 13.79	39.9 ± 0.1	—	—
W409F	0.45	3.25 ± 0.21	595.0 ± 9.6	182.86 ± 12.29	39.3 ± 0.2	—	1.2
W409G	0.44	3.85 ± 0.82	14.1 ± 0.7	3.66 ± 0.81	—	—	—	³
W409H	2.87	2.34 ± 0.26	106.8 ± 2.8	45.54 ± 5.17	39.3 ± 0.2	—	2.1
W409H	0.58	2.60 ± 0.32	616.4 ± 18.0	237.36 ± 29.96	38.5 ± 0.4	—	2.2
W409H	0.72	3.16 ± 0.19	402.2 ± 5.9	127.29 ± 7.96	38.9 ± 0.9	—	2.1
W409I	1.13	4.64 ± 0.50	6.1 ± 0.2	1.32 ± 0.15	40.3 ± 0.3	—	10.7
W409I	1.12	9.11 ± 1.79	744.7 ± 45.1	81.71 ± 16.81	40.3 ± 0.2	—	5.7
W409K	0.48	1.62 ± 0.22	180.6 ± 5.4	111.29 ± 15.53	38.5 ± 0.3	—	3.5
W409L	6.10	2.05 ± 0.16	87.5 ± 1.6	42.57 ± 3.43	39.5 ± 0.3	—	9.5
W409M	0.62	7.11 ± 2.14	35.4 ± 2.6	4.98 ± 1.55	40.0 ± 0.1	—	-0.6
W409N	4.93	8.43 ± 1.13	298.7 ± 12.0	35.42 ± 4.97	—	38.9 ± 0.1	1.9
W409N	8.27	3.38 ± 0.55	69.3 ± 2.8	20.50 ± 3.45	39.1 ± 0.5	—	2.0
W409P	—	—	—	—	—	—	82.7
W409R	3.39	2.72 ± 0.34	90.3 ± 2.7	33.16 ± 4.27	—	42.0 ± 0.3	2.6
W409R	1.95	2.74 ± 0.30	194.6 ± 5.1	70.98 ± 7.92	—	47.0 ± 0.3	4.8
W409R	0.74	1.98 ± 0.11	74.7 ± 0.9	37.80 ± 2.11	34.6 ± 0.6	—	—
W409S	6.17	4.52 ± 0.76	143.7 ± 6.3	31.79 ± 5.51	39.4 ± 0.4	—	4.1
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
W409T	1.78	2.08 ± 0.51	344.1 ± 19.4	165.50 ± 41.39	37.1 ± 0.3	—	3.1
W409T	0.12	5.29 ± 1.08	85.2 ± 4.7	16.11 ± 3.42	35.8 ± 1.7	—	-716.9
W409V	4.17	9.62 ± 1.08	139.5 ± 4.8	14.50 ± 1.71	39.3 ± 0.9	—	5.0
W409Y	*	2.60 ± 0.18	662.4 ± 10.8	254.80 ± 18.18	36.8 ± 0.1	43.5 ± 0.0	—	²
W409Y	0.26	2.99 ± 0.36	725.2 ± 26.7	242.89 ± 30.82	—	—	0.4
W409Y	1.45	3.34 ± 0.23	513.6 ± 8.6	153.86 ± 10.77	40.0 ± 0.5	—	0.4
Y411R	0.15	1.24 ± 0.36	62.8 ± 3.9	50.73 ± 15.21	—	—	—
Y411R	0.41	2.74 ± 0.81	37.8 ± 2.6	13.81 ± 4.19	—	—	—
K413A	3.15	7.32 ± 0.60	381.9 ± 9.0	52.18 ± 4.42	—	48.8 ± 0.1	—
K413Q	3.02	5.41 ± 0.62	386.6 ± 12.1	71.53 ± 8.55	—	45.8 ± 1.7	5.0
K413R	0.41	0.85 ± 0.29	470.8 ± 32.0	555.04 ± 196.18	40.1 ± 0.1	—	—
R414K	0.55	26.88 ± 10.87	399.9 ± 66.3	14.88 ± 6.50	40.6 ± 0.6	—	1.6
F415A	*	16.63 ± 4.00	—	—	40.5 ± 0.3	44.7 ± 0.0	—	²
F415H	0.47	0.78 ± 0.34	9.2 ± 1.0	11.81 ± 5.23	—	—	2.3
F415N	*	—	—	—	—	—	—	²
F415Y	2.24	10.01 ± 1.13	288.3 ± 9.8	28.82 ± 3.40	—	—	—	³
F415Y	1.98	8.05 ± 0.82	116.3 ± 3.5	14.46 ± 1.53	—	41.6 ± 0.2	—
I417H	1.25	2.81 ± 0.87	4.2 ± 0.3	1.51 ± 0.48	—	44.5 ± 0.8	8.6
H419Y	3.23	5.90 ± 0.19	1,097.6 ± 13.2	185.88 ± 6.29	—	—	—	³
H419Y	3.32	3.86 ± 0.59	387.7 ± 14.9	100.45 ± 15.74	—	49.1 ± 1.1	-2.1
N421D	0.57	4.12 ± 0.23	1,478.0 ± 21.9	358.47 ± 20.52	41.7 ± 0.2	—	-0.2
N421D	0.69	3.54 ± 0.44	873.3 ± 27.0	246.82 ± 31.41	41.4 ± 0.2	—	-0.2
N421D	1.12	2.13 ± 0.29	988.6 ± 28.6	463.34 ± 63.67	40.8 ± 0.1	—	-0.2
Y422F	2.83	30.66 ± 5.28	183.8 ± 13.5	6.00 ± 1.12	—	47.4 ± 0.2	-0.4
E423S	*	6.60 ± 0.42	530.4 ± 34.6	80.31 ± 7.36	39.5 ± 0.1	44.8 ± 0.0	—	²
Variant	Yield, c_E (mg/mL)	K_M (mm)	k_cat (min⁻¹)	k_cat/K_M (mm⁻¹min⁻¹)	T₅₀ (°C)	T_m (°C)	Rosetta score change
T424H	3.64	6.10 ± 0.34	39.6 ± 0.6	6.50 ± 0.37	—	46.7 ± 0.2	1.7
R427H	0.46	1.28 ± 0.41	1.0 ± 0.1	0.80 ± 0.26	—	—	11.6
R427H	1.00	3.69 ± 0.52	7.2 ± 0.3	1.95 ± 0.28	—	44.3 ± 0.3	12.0
R427L	0.41	4.95 ± 0.40	380.1 ± 8.3	76.73 ± 6.49	—	45.4 ± 0.3	4.9
R427L	0.41	4.95 ± 0.40	380.1 ± 8.3	76.73 ± 6.49	—	45.4 ± 0.3	4.9
R427L	2.20	4.22 ± 0.32	371.6 ± 7.3	88.01 ± 6.94	—	43.4 ± 0.0	—
R427W	0.31	0.98 ± 0.27	35.6 ± 2.0	36.11 ± 10.00	40.2 ± 0.1	—	—
P429A	2.51	9.81 ± 1.03	327.2 ± 10.7	33.35 ± 3.65	—	—	—
L434S	2.49	0.57 ± 0.16	239.0 ± 13.1	416.14 ± 121.04	—	43.2 ± 0.0	1.2
L434S	0.97	2.81 ± 0.20	204.8 ± 3.5	72.87 ± 5.31	—	42.7 ± 0.0	1.2
W435Y	0.48	7.41 ± 0.72	823.9 ± 23.2	111.17 ± 11.22	—	—	—
W435Y	*	—	—	—	40.2 ± 0.2	—	—
A441V	2.07	4.65 ± 0.10	859.5 ± 4.6	184.98 ± 4.01	—	—	—	³