4.3. Substrate Specificity Analysis

The specificity of RmCE towards different monosaccharides was determined in MOPS buffer (pH 6.3) and with 200 mM of substrate (d-glucose, d-mannose, d-galactose, d-xylose, d-lyxose, l-arabinose) at an enzyme concentration of 0.3 mg/mL. All reactions were monitored at 70 °C and 5 time points were taken during 24 h. Enzyme inactivation was achieved by transferring the sample (5 μL) in 100 mM of NaOH (90 μL). Conversion of substrate to product was evaluated by high-performance anion exchange chromatography–pulsed amperometric detection (HPAEC–PAD) using the Dionex ICS-3000 system (Thermo Fischer Scientific) (CarboPac PA20 column-3 × 150 mm) as described by Verhaeghe et al. but with a 15-min isocratic method of 20 mM NaOH [40]. The monosaccharides were quantified by determining conversion based on the peak areas. Product concentration was then plotted in function of time and a linear correlation was fitted representing the activity of the enzyme (in μmol/min). Finally, specific activities were calculated by dividing the increase in product (units) by the enzyme concentration (mg). One unit (U) of RmCE activity was defined as the amount of enzyme required to produce 1 μmol of product from substrate per minute at 70 °C and pH 6.3.