Standard grade glass capillaries

Enzyme melting temperature (T_m) was estimated at different pH ranging 4–9, as well as pH 6.5 with 1 percent (w/v) WAX, using nanoDSF applying a Prometheus NT.48 instrument (NanoTemper Technologies). Standard grade glass capillaries (NanoTemper Technologies) were filled with enzyme solution at a concentration of 0.1–0.2 mg/mL. Thermal unfolding was performed with a temperature gradient between 20 and 95°C at a 1°C/min ramp rate and adjusting excitation power to 60 percent. T_m was determined from the first derivative of the absorbance ratio 350/330 nm and was identified automatically by the instrument software PR.ThermControl (NanoTemper Technologies).
HhXyn5A thermostability and inactivation over time was investigated by incubating the enzyme at 50 and 60°C for 24 h while monitoring the retained reaction rate by removing aliquots of enzyme over time and immediately chilling aliquots on ice. Retained activity was then evaluated in a 10 min reaction using 10 mg/mL WAX at 50°C with the DNS assay performed in a micro scale incubating thermocycler with heat gradient mode measuring absorbance using with Multiskan GO microplate spectrophotometer.

Thermal stability of purified XylT1, β4GalT7, SUMO-β3GalT6, and GlcAT-1 were assessed using nano differential scanning fluorimetry technology (nanoDSF). Protein samples were diluted to a concentration of 0.3 mg/mL in their corresponding storage buffers: XylT1 and SUMO-β3GalT6 in Tris A buffer with 5% glycerol (v/v), β4GalT7 in S200 buffer, and GlcAT-1 in HEPES A buffer. Standard-grade glass capillaries (Nanotemper) were filled with 15 μL of protein suspension and loaded onto the tray of Prometheus NT.48 instrument (NanoTemper Technologies). The experiment was run using a 1.5 °C per minute thermal ramp over a temperature range from 25 °C to 95 °C at 80%–100% excitation power. Acquisition was followed by data analysis using the PR.ThermControl v.2.3.1 software in order to establish melting temperatures for each sample.