Cosmosil 5c18 ar 2 packed column

Aspergillus sp. OPMF00815, grown on LCA agar plates, was inoculated in modified potato dextrose broth (mPDB) (PDB with KNO₃, KH₂PO₄, Mg₂SO₄, and artificial sea water) and incubated at 28°C for 7 days at 250 rpm. Aliquots of pre-cultured cells were further inoculated into fresh mPDB and incubated at 28°C for 7 days at 250 rpm. The culture medium was centrifuged at 2,850 × g for 10 min. The supernatant of the culture medium was extracted with ethyl acetate, condensed, and fractionated through a stepwise gradient of methanol (0%, 20%, 40%, 60%, 80%, and 100%) using a Purif-Pack column (ODS, size: 20) (Shoko Science). After stepwise purification with methanol, the samples were repeatedly purified using an HPLC (Shimadzu) equipped with a COSMOSIL 5C18-AR-II packed column (φ20 × 250 mm) (Nacalai Tesque). The mobile phase consisted of solvent A (0.1% formic acid) and solvent B (0.1% formic acid-acetonitrile), and the isocratic elution with 17% solvent B was performed at a flow rate of 10 mL/min and monitored at 210 nm. The cell pellets were treated with acetone and methanol and further treated with ethyl acetate as described above. The samples treated with ethyl acetate were fractionated through a stepwise gradient of methanol and subjected to repeated purification by HPLC, as described above.

The activity of EhGalE was examined on a Reversed Phase HPLC (Shimadzu, Japan) using a COSMOSIL 5C18-AR-II packed column (Nacalai Tesque, Japan) at RT with absorbance set at 254 nm to detect nucleotide sugars. The HPLC gradient was based on buffer A (100 mM potassium phosphate pH 6.4 supplemented with 8 mM tetrabutylammonium hydrogen sulfate in HPLC grade H₂O) and Buffer B (70% buffer A and 30% acetonitrile) as follows: 100% buffer A for 13 min; 0–77% linear gradient of buffer B for 22 min; 77–100% buffer B for 1 min; and 100% buffer B for 14 min using a constant flow rate of 0.8 ml/min [62 (link)]. Peaks were identified by comparison with the retention times of a standard mixture injected before the analysis. Each nucleotide sugar peak was integrated and quantified based on the peak areas of the calibration curve of each standard and was further identified using MALDI-ToF MS. Retention times were standardised against NAD⁺ peaks when a small shift was observed.