Table 3 Properties of wtPTE InDel variants selected for improved arylesterase activity.

From: Accessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis

 

AE

PTE

Thermal denaturation

PTE varianta

kcat (s−1)

KM (µM)

kcat/KM (M−1 s−1)

kcat (s−1)

KM (µM)

kcat/KM (M−1 s−1)

Tm (°C)d

wtPTEb

0.075 ± 0.004

179 ± 21

(4.2 ± 0.6) × 102

1270 ± 27

57 ± 5

(2.2 ± 0.2) × 107

78.1 ± 0.2

H254Rb

0.27 ± 0.01

250 ± 32

(1.1 ± 0.2) × 103

62 ± 3

7 ± 1

(8.9 ± 1.4) × 106

88 ± 0.1

∆A270L271L272G273c

2.1 ± 0.1

258 ± 40

(8.2 ± 1.4) × 103

55 ± 4

148 ± 22

(3.7 ± 0.7) × 105

82 ± 1

P256R/G256aA256bc

4.6 ± 0.3

381 ± 59

(1.2 ± 0.3) × 104

54 ± 3

988 ± 114

(5.4 ± 0.7) × 104

84.3 ± 0.3

S256aG256bc

8.9 ± 0.7

821 ± 152

(1.1 ± 0.3) × 104

13 ± 1

116 ± 21

(1.1 ± 0.3) × 105

77.5 ± 0.4

G311ac

4.2 ± 0.2

292 ± 34

(1.5 ± 0.2) × 104

39 ± 1

307 ± 25

(1.3 ± 0.2) × 105

75.2 ± 0.3

  1. AE, arylesterase (substrate: 2-NH); PTE, phosphotriesterase (substrate: paraoxon).
  2. aThe symbol ∆ before a residue (or a group of residues) signifies that this (or these) residue(s) have been deleted. Inserted residues are labelled using the number of the position after which they are inserted and alphabetical order (e.g., glutamine and tyrosine residues inserted in this order after the residues at position 230 would be labelled Q230aY230b).
  3. bKinetic parameters are from Tokuriki et al.18.
  4. cSee Supplementary Fig. S15 for detailed experimental conditions for Michaelis–Menten kinetics.
  5. dThermal denaturation for wtPTE and all InDel variants was measured with SYPRO Orange as the fluorescent probe and Tm is given as mean ± standard deviation (from six or more measurements; Supplementary Fig. S16). For variant H254R, the Tm value is from Wyganowki et al.61.
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