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Table 2 Kinetic parameters of the alkaline serine protease DHAP and its variants at different temperaturesa

From: Engineering Bacillus pumilus alkaline serine protease to increase its low-temperature proteolytic activity by directed evolution

Variants

15 °C

50 °C

Km (mM)

k cat

kcat/Km

(s− 1·M− 1)

Fold

()

Km (mM)

k cat

kcat/Km

(s− 1·M− 1)

Fold

()

(s−1)

(s−1)

wt

0.16 ± 0.02

102 ± 03

0.64E × 106

1.00

0.23 ± 0.018

609 ± 14

2.65E × 106

1.00

P9S

0.41 ± 0.09

362 ± 43

0.88E × 106

1.37

0.43 ± 0.045

829 ± 81

1.97E × 106

0.74

A1G/K27Q

0.20 ± 0.04

373 ± 27

1.87E × 106

2.92

0.36 ± 0.081

1719 ± 161

4.78E × 106

1.83

A38V

0.13 ± 0.02

176 ± 11

1.35E × 106

2.11

0.20 ± 0.096

952 ± 123

4.76E × 106

1.79

A116T

0.22 ± 0.05

262 ± 22

1.19E × 106

1.86

0.25 ± 0.040

778 ± 71

3.11E × 106

1.17

T162I

0.17 ± 0.03

300 ± 20

1.76E × 106

2.75

0.26 ± 0.026

1149 ± 53

4.41E × 106

1.66

S182R

0.19 ± 0.03

259 ± 18

1.36E × 106

2.13

0.23 ± 0.50

897 ± 102

3.90E × 106

1.47

T351S

0.31 ± 0.05

283 ± 24

0.91E × 106

1.42

0.25 ± 0.053

989 ± 97

3.96E × 106

1.49

P9S/T162I

0.30 ± 0.013

450 ± 10.2

1.50E × 106

2.34

0.26 ± 0.009

1169 ± 22

4.50E × 106

1.70

P9S/K27Q

0.35 ± 0.035

645 ± 28.5

1.84E × 106

2.88

0.32 ± 0.015

1555 ± 40

4.86E × 106

1.83

K27Q/T162I

0.21 ± 0.013

288 ± 5.9

1.37E × 106

2.14

0.25 ± 0.018

1092 ± 22

4.37E × 106

1.65

P9S/K27Q/T162I

0.34 ± 0.016

579 ± 19.9

1.70E × 106

2.65

0.34 ± 0.016

1596 ± 47

4.69E × 106

1.77

  1. aThe hydrolytic reaction was performed in 20 mM Tris-HCl buffer (pH 8.0) containing 2 mM CaCl2 with 0.05–1.2 mM AAPF-pN as substrate and 50 ng (at 15 °C) or 25 ng (at 50 °C) of enzyme