Table 3 Enzyme kinetics and stability of selected variants.

From: Two-substrate enzyme engineering using small libraries that combine the substrate preferences from two different variant lineages

Variant

Mutations

Vm (mM/min)

kcat (/s)

Km (mM)

Specific activity

at 50 mM 3-FBA

(µmol/mg min)

Conversion @24 h

(%)

Tm (°C)

WT

 

0

0

n.d.

0

0

57.8

6Ma

4M+H100L+D469T

0.012

0.22 (0.01)

n.d.

0.048 (0.005)

2.5

n.d.

TK-1

6M+S385Y

11 (7)

134 (85)

220 (180)

20.3 (5)

61

59.2

TK-2

6M+R520Q/S385Y

9.7 (3)

118 (41)

530 (190)

8.3 (0.3)

61

61.4

TK-3

6M+S385F

3.5 (0.2)

43 (3)

82 (11)

13.3 (1.5)

62

65.1

TK-5C

6M+R520Q/S385pCNF

4.2 (1.1)

51 (13)

95 (45)

14.6 (3)

62

63.7

  1. aKinetic parameters for 6M (H100L/H192P/A282P/I365L/D469T/G506A) were published previously4, using 0.067 mg/mL enzyme and by varying pyruvate at 50 mM 3-FBA. Km is for 3-FBA at 50 mM pyruvate. Errors (as SEM) are shown in parentheses.
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