The optimal pH of xylanase was measured at 37 °C using 50.00 mM buffer with pH values ranging from 2.0 to 12.0, including Glycine-HCl (Gly-HCl) buffer (pH 2.0–3.0), citrate buffer (pH 3.0–6.5), phosphate buffer (pH 6.5–8.0), barbital sodium buffer (pH 8.0–9.5), Glycine-NaOH (Gly-NaOH) buffer (pH 9.5–10.5), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) buffer (pH 10.5–11.0), and NaH2PO4-NaOH buffer (pH 11.0–12.0). The pH stability of xylanase was determined by incubating the enzyme in buffers with pH ranging from 2.0 to 12.0 for 30 min at 37 °C, and then measuring the residual enzyme activity. The optimal temperature of xylanase was detected for the enzyme activity in 50 mM citrate buffer at temperatures ranging from 30 °C to 80 °C. The thermostability of xylanase was determined by incubating the enzyme at temperatures ranging from 30 °C to 70 °C for 30 min in 50.00 mM citrate buffer.
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 inSection 2.4 . The kinetic parameters of xylanase were measured with different concentrations (2.50–30.00 mg/mL) of beechwood xylan in optimal reaction conditions. The values of Km and Vmax of the enzyme were calculated according to the Michaelis-Menten equation using GraphPad Prism software [40 (link),52 (link)].
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
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