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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