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).