Table 1 Hydrolytic kinetic resolution of different substrates using (S)-selective and (R)-selective AFEST mutants

From: Efficient molecular evolution to generate enantioselective enzymes using a dual-channel microfluidic droplet screening platform

Substrate

Enzyme

Reaction time (min)

Conv. (%)

eep (%)

E

rac-1

WT

10

23

70 (R)

7.4 (R)

rac-1

6A8

10

20

>99 (S)

>100 (S)

rac-1

4D11

6

18

97 (S)

80 (S)

rac-1

4E12

20

47

95 (R)

74 (R)

rac-2

WT

20

22

40 (R)

3.0 (R)

rac-2

6A8

30

24

79 (S)

11 (S)

rac-2

4D11

10

21

94 (S)

35 (S)

rac-2

4E12

20

26

82 (R)

13 (R)

rac-3

WT

20

18

81 (R)

12 (R)

rac-3

6A8

20

22

92 (S)

31 (S)

rac-3

4D11

10

34

93 (S)

42 (S)

rac-3

4E12

20

38

75 (R)

11 (R)

rac-4

WT

6

27

37 (R)

3.0 (R)

rac-4

6A8

60

29

68 (S)

7.4 (S)

rac-4

4D11

3

25

95 (S)

88 (S)

rac-4

4E12

20

22

68 (R)

6.7 (R)

rac-5

WT

10

24

46 (R)

3.6 (R)

rac-5

6A8

60

18

78 (S)

10 (S)

rac-5

4D11

3

25

95 (S)

52 (S)

rac-5

4E12

10

26

64 (R)

6.2 (R)

  1. Note: The reactions were performed at 37 °C with 10 μg enzyme and 100 μM substrate in a total reaction volume of 500 μL. The reaction products were detected by HPLC equipped with a chiral column (Chiralcel OJ-H, Daicel). The mobile phases used were n-hexane containing 0.1% vol vol−1 TFA and isopropanol at different ratios (90:10 for rac-1; 85:15 for rac-2, rac-3; and 95:5 for rac-4, rac-5). The total flow rate of mobile phases was 1 mL min−1
  2. AFEST Archaeoglobus fulgidus esterase, HPLC high-performance liquid chromatography, TFA trifluoroacetic acid