Pa1 analytical column

High-performance
anion
exchange chromatography with pulsed amperometric detection (HPAEC-PAD)
and matrix-assisted laser desorption ionization–time-of-flight
mass spectrometry (MALDI-ToF MS) were used to analyze oxidized products.
HPAEC-PAD was conducted using a Dionex ICS5000 system equipped with
a CarboPac PA1 analytical column (2 × 250 mm) and a CarboPac
PA1 guard column (2 × 50 mm). Product separation was achieved
by applying a 29 min gradient as previously described for cello-oligosaccharides
(Figure S3).^{47 (link)} Oxidized products were quantified by using in-house made standards
as described elsewhere.^{46 (link)} Chromatograms
were recorded and analyzed with Chromeleon and plot preparation was
done in Microsoft Excel. MALDI-ToF MS was performed on an Ultraflex
MALDI-ToF/ToF instrument (Bruker Daltonik GmbH, Bremen, Germany) equipped
with a Nitrogen 337 nm laser, as described previously.^{18 (link)}

Filtrates of the reactions were analyzed by HPAEC–PAD on a Dionex ICS-5000 system (Sunnyvale, CA, USA) equipped with a CarboPac PA1 guard column (2 × 50 mm) and a CarboPac PA1 analytical column (2 × 250 mm). The flow was set at 0.25 mL/min, with an injection volume of 10 µL. For elution, two mobile phases were used: 0.1-M NaOH as eluent A, 1-M NaOAc in 0.1-M NaOH as eluent B. The following gradient was applied: linear increase from 0 to 10% B in 10 min, then to 30% B in the next 15 min, followed by an exponential gradient to 100% B in 5 min. The column was reconditioned before every injection by running 0% B for 9 min. Assignments of C1- and C4-oxidized peaks was based on comparison with products released from reference enzymes performing either C1- or C4-oxidation (NcLPMO9F and NcLPMO9C, respectively), which were produced as described elsewhere [45 (link)]. Gluconic acid and cellobionic acid were assigned and quantified using available standards (purchased from Megazyme and Synthose, respectively). To analyze the chromatograms, Chromeleon version 7.0 software (Thermo Fisher Scientific, Waltham, MA, USA) was used.