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Fig. 6 | Microbial Cell Factories

Fig. 6

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

Fig. 6

Enzymatic stability for wild type SMO and its variants in E. coli expression host. Thermal stabilities of the enzymes were carried out by purified enzyme incubation in KP (potassium phosphate) buffer (200 mM, pH 8.0) for 12 h at a range of temperatures from 30 to 65 °C. Afterwards, the residual activity of the pre-incubated SMOs was tested in the following reaction system: 0.8 U/mL purified SMOA, 1.6 U/mL of purified SMOB, 1.7 U/mL formate dehydrogenase, 0.2 M sodium formate, 0.3 mM NADH, 1 mM NAD+, 0.05 mM FAD, and 200 mM styrene (from a 200-fold stock in ethanol). And the pH stability of the wild type and variants was determined by purified enzyme incubation at 30 °C for 12 h in the following buffer system: 0.05 M acetate buffer (pH 3.0–6.0), 0.05 M phosphate buffer (pH 6.0–8.0), and 0.05 M glycine–NaOH buffer (pH 8.0–11.0). After incubation, their residual enzyme activities were measured at 30 °C in KP buffer (200 mM, pH 8.0). a Enzyme inactivation assay at different temperatures for 12 h. b Time courses of thermal inactivation at 60 °C. c Enzyme inactivation assay at different pH for 12 h. The initial activity before incubation was set to 100%, 100%-value corresponds to an initial activity of 5.6 ± 0.21 U/mg. All assays were performed in triplicate and the standard deviations of the biological replicates are represented by error bars

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