The protein concentration of the pure enzyme solution was diluted to 0.50 mg/mL using citrate buffer at pH 6.0, and the diluted enzyme solutions of the mutant enzyme and XynA were kept at 60 °C for 0, 15, 30, 60, 90, 120 and 180 min, respectively, and cooled rapidly in an ice water bath. The activity of untreated xylanase was defined as 100%, and the residual enzyme activity was calculated using the formula y = Ae−kt (A is the initial enzyme activity, t is time, k is the decay constant). The half-life t1/2 is equal to ln2/k. The half-lives of mutants and XynA at 60 °C were calculated separately according to the formula [49 (link)]. The Protein Thermal Shift ™ dye kit was used to determine the thermal denaturation temperature of mutants and XynA [50 (link)]. Quantitative real-time PCR was performed using the CFX Touch 96-well system (Bio-Rad, California, CA, USA). Samples were heated on a 0.05 °C/s gradient from 4 to 95 °C and protein unfolding at each temperature was monitored by measurement of fluorescence at 580/623 nm (excitation/emission) [51 (link)].
Substrate specificity of the enzymes was investigated using the standard assay procedure with one of the following substrates (1%, w/v), beechwood xylan, birchwood xylan, or oat-spelt xylan. The liberated reducing sugars’ concentrations were determined by the DNS method, as described in