The optimum temperature was determined as follows. Equal amounts of purified wild-type and variants were taken, and the substrate concentration was set at 12 mM for p-nitrobenzaldehyde and 48 mM for nitromethane. Reactions were carried out at 20 °C, 30 °C, 40 °C, 50 °C, 60 °C, and 70 °C.
To determine the thermal stability of wild-type at different temperatures, equal amounts of purified WT were placed in water baths at temperatures of 30 °C, 40 °C, 50 °C, and 60 °C. At regular intervals, enzyme samples were taken for reaction, with the initial enzyme activity set at 100%.
To determine the thermal stability of wild-type and different variants at 60 °C, equal amounts of purified wild-type and variants were incubated at 60 °C. At regular intervals, enzyme samples were taken for reaction, with the initial enzyme activity set at 100%.
The optimum pH was determined as follows. Equal amounts of purified wild-type and variant were taken, and the substrate concentration was set at 12 mM for p-nitrobenzaldehyde and 48 mM for nitromethane. Experiments were conducted at a temperature of 40 °C in different buffer solutions at pH 6, 7, 7.5, 8, 8.5, and 9.
The kinetic parameters of wild-type enzyme and variants were determined by measuring the reaction rate with different concentrations of p-nitrobenzaldehyde from 3 to 18 mM used for the reaction, while the concentration of nitromethane was set as excess at 1 M. The purified enzyme was added at a final concentration of 0.3 mg/mL and reactions were carried out at 40 °C and 750 rpm for 5 min. The reaction rates of LmrR at different substrate p-nitrobenzaldehyde concentrations were determined. The kinetics parameters were determined using the Michaelis−Menten equation.
To determine the thermal stability of wild-type at different temperatures, equal amounts of purified WT were placed in water baths at temperatures of 30 °C, 40 °C, 50 °C, and 60 °C. At regular intervals, enzyme samples were taken for reaction, with the initial enzyme activity set at 100%.
To determine the thermal stability of wild-type and different variants at 60 °C, equal amounts of purified wild-type and variants were incubated at 60 °C. At regular intervals, enzyme samples were taken for reaction, with the initial enzyme activity set at 100%.
The optimum pH was determined as follows. Equal amounts of purified wild-type and variant were taken, and the substrate concentration was set at 12 mM for p-nitrobenzaldehyde and 48 mM for nitromethane. Experiments were conducted at a temperature of 40 °C in different buffer solutions at pH 6, 7, 7.5, 8, 8.5, and 9.
The kinetic parameters of wild-type enzyme and variants were determined by measuring the reaction rate with different concentrations of p-nitrobenzaldehyde from 3 to 18 mM used for the reaction, while the concentration of nitromethane was set as excess at 1 M. The purified enzyme was added at a final concentration of 0.3 mg/mL and reactions were carried out at 40 °C and 750 rpm for 5 min. The reaction rates of LmrR at different substrate p-nitrobenzaldehyde concentrations were determined. The kinetics parameters were determined using the Michaelis−Menten equation.
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