3.4 Measurement of catalytic activity
Enzyme expression and purification of r-Rha1 mutants, the α-L-rhamnosidase activity follows the same protocol in the previous study.56 For kinetic experiments, five different concentrations (within 2-6 mM) of p NPR were used to incubate the purified α-L-rhamnosidase at 60ºC and pH 4.0 (20 mM of citrate acid buffer) for 10 min. The K m andV max were calculated from Lineweaver-Burk plots.57 These values were further used to calculate the k cat and the catalytic efficiency.
3.5 Measurement ofthermostability
The optimal temperatures for wild type and mutants were determined by measuring the enzyme activities at different temperatures (ranging from 30 to 80 °C) in 20mM citrate acid buffer (pH 4.0) with p NPR as the substrate. The thermostability was estimated by measuring the ratio of the residual activity to the initial activity of the enzyme. Samples were diluted in 20mM buffer (pH 4.0) to a protein concentration of 0.7 mg/mL followed by incubating at 60 °C, 65 °C, and 70 °C, respectively. After heat treatment, the samples were cooled on ice immediately. Relative activity was estimated with original activity taken as 100 %.
3.6 Circular dichroism spectroscopy analysis
Circular dichroism (CD) measurements were recorded from 190 to 260 nm at 25°C with a Chirascan Circular Dichroism spectrometer (Applied Photophysics, UK) at a scan rate of 100 nm/min, 0.25 s of interval and 1nm of bandwidth for identifying secondary structure. CD measurements were recorded from 190 to 260 nm at 20-100°C to determination of heat distortion temperature (Tm).