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Table 1 The kinetic parameters of mutants screened from site-saturation mutagenesis at position 305 compared to the wild type

From: Asp305Gly mutation improved the activity and stability of the styrene monooxygenase for efficient epoxide production in Pseudomonas putida KT2440

Mutations Km (mM) kcat (s−1) kcat/Km (mM−1 s−1)
Wild type 0.85 ± 0.12 1.08 ± 0.03 1.27 ± 0.15
D305V 0.54 ± 0.08 1.43 ± 0.03 2.65 ± 0.17
D305A 0.46 ± 0.11 1.53 ± 0.02 3.33 ± 0.21
D305G 0.36 ± 0.14 1.70 ± 0.02 4.72 ± 0.33
  1. The reaction was carried out in 2 mL volumes containing 0.2 M KP (potassium phosphate) buffer (pH 8.0), 0.8 U/mL purified SMOA, 1.6 U/mL of purified SMOB, 1.7 U/mL formate dehydrogenase (FDH; EC 1.2.1.2, from Candida boidinii), 0.2 M sodium formate, 0.3 mM NADH, 1 mM NAD+, 0.05 mM FAD, and varying concentrations of styrene (from a 200-fold stock in ethanol). All enzymes were previously expressed in E. coli BL21. The reaction mixture was shaken at 200 rpm and 30 °C for 1 h, and then it was extracted with ether and analyzed with reverse phase HPLC on a Luna C18 (4.6 mm × 150 mm) column at a flow rate of 0.8 mL/min