Carbopac pa 100

Whey permeate, mother liquor, diafiltered oligosaccharides-enriched retentate and milk oligosaccharides-rich powder, appropriately diluted in water, were analyzed for quantification of lactose and sialyllactose.
Lactose was quantified by HPLC using an HPX-87C Carbohydrate column (300×7.8 mm) (Aminex, Bio-Rad, UK) and a refractive index detector. The elution was obtained in isocratic conditions using 9 mM sulphuric acid for 30 min.
Both sialyllactose isomers (3′-sialyllactose and 6′-sialyllactose) were quantified by High pH Anion Exchange Chromatography with Pulsed Amperometric Detection, using a CarboPac PA 100 (250×4 mm) connected to a CarboPac PA 100 guard column and equipped with an electrochemical detector (Dionex Corporation, Sunnyvale, CA). Elution was carried out with the following gradient: 100 mM NaOH (Eluent A) and 100 mM NaOH, 500 mM NaAc (Eluent B) (t = 0–3 min 95% Eluent A; t = 3–13 min 88% Eluent A; t = 13–30 min 50% Eluent A; t = 30–45 min equilibrated at 95% Eluent A).

Pro was extracted from 50 mg of ground lettuce leaf with 100 µL water and subsequently sonicated for 10 min. Samples were then centrifuged at 13,000× g for 5 min. The supernatant was diluted 10 times in water containing 20 µM serine (used as internal standard). The sample was then vortexed and centrifuged at 13,000× g for 5 min, transferred to a vial with a conical glass insert and analyzed by HPAEC-IPAD (Thermofisher, USA) in order to determine the Pro content after separation on a CarboPac^® PA100 anion exchange column. The flow rate was 0.25 mL·min⁻¹. Pro was eluted with the following gradient: 9 mM NaOH from 0 min to 15 min; 0.5 M NaAc from 15 to 17 min; 90 mM NaOH from 17 to 23 min. Quantification was performed on the peak areas with the external standard, consisting of 20 µM Pro.

The total reducing sugar liberated from enzymatic hydrolysis was measured using the 3,5-dinitrosalicylic acid (DNS) assay [44 (link)] with glucose as the standard. The hydrolysates of (hemi)cellulosic substrates were separated and quantified using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD; ThermoFisher, Sunnyvale, CA) equipped with a CarboPac™ PA-100 (4 mm × 250 mm) column. A multi-step gradient was performed to analyze the hydrolysates of CMC and lichenan using the previous method [45 (link)]. The substances CE1, CE2, CE3, CE4, CE5, and CE6 were used as the standards. To analyze the hydrolysates of mannohexaose, the eluents were deionized water (eluent A) and 0.1 M sodium hydroxide (eluent B) at a flow rate of 0.45 mL/min, using the multi-step procedure as follows: 0–4 min, isocratic, 20% B; 4–5 min, linear, 20%–100% B; 5–25 min, isocratic, 100% B; 25–28 min, linear, 100%–20% B; and 28–31 min, isocratic, 20% B. The substances M1, M2, M3, M4, M5, and M6 were used as the standards. The hydrolysates of raw material substrate were analyzed by ultra-high-performance liquid chromatography–high-resolution mass spectrometry (UHPLC–HRMS; TripleTOF™ 5600 + , AB SCIEX, USA) with Poroshell 120 EC-C8 (100 mm × 4.6 mm, 4 μm, Agilent) as described previously [45 (link)].

Enzyme reaction products and substrate (LRP alkali) were analyzed by HPAEC-PAD using a CarboPac PA100 (4.6 × 250 mm) and guard (4.6 × 50 mm) on a Dionex ICS3000 system (Thermo Fischer Scientific, Sunnyvale, CA, USA). The column was operated at 1 mL/min with eluent A (water), eluent B (500 mM NaOH), and eluent C (500 mM sodium acetate) according to the following gradient: 0–2 min isocratic 40% B and 1% C, 2–35 min linear gradient to 40% B and 45% C, hereafter immediately shifted to 5% B and 90% C and these conditions were kept for 4 min and ended by shifting back to starting conditions and reconditioning for 6 min.

The activity of MtLPMO9G, MtLPMO9G-CDL, and MtLPMO9G-CD was evaluated using PASC as the substrate. The reaction mixture contained 50 mM NH₄Ac (pH 5), 1 μM LPMO, 1 mM AscA, and 4 mg mL⁻¹ PASC, and the reaction proceeded for 24 h at 45 °C with continuous shaking at 1000 rpm in an Eppendorf Thermomixer. The resulting products were analyzed by HPAEC using an ICS3000 system equipped with a pulsed amperometric detector (Thermo Fisher Scientific) and an analytical CarboPac PA-100 (4 × 250) as previously described (33 (link), 34 (link)). C1-oxidized cello-oligosaccharide standards for the HPAEC-pulsed amperometric detector were prepared by oxidizing pure cello-oligosaccharides (Megazyme) with iodine as described earlier for the preparation of cellobionic acid (54 (link)). Additionally, the products were hydrolyzed by cellobiohydrolase Ⅰ(CBH Ⅰ) (Megazyme) and quantitatively analyzed by HPAEC.

The ability of BT4656^S1_11 to remove O6-linked sulfate from GlcNAc6S was monitored by the production of GlcNAc detected by high-performance anion exchange chromatography (HPAEC) with PAD using a carbohydrate standard quad waveform for electrochemical detection at a gold working electrode with an Ag/AgCl pH reference electrode and a Carbopac PA-100 guard and analytical column (Dionex; ThermoFisher). Assays were performed in 1 ml volumes. After first removing a zero time point sample, enzyme was added, then 110 μl volumes were removed, and boiled to destroy enzyme activity, at periodic time intervals over 18 min (Figure 2E,F). Samples were then centrifuged and subjected to HPAEC. GlcNAc was separated using an isocratic gradient ran over 20 min with 100 mM NaOH, the column was then stripped and washed with 10 min runs of 100 mM NaOH plus 500 mM Sodium acetate then 10 min with 500 mM NaOH before being ran back into 100 mM NaOH ready for the next sample. A flow rate of 1.0 ml/min was used throughout.