Hypersil ods

The substrate specificity of the mussel (C. gigas) and snail (A. vulgaris) β-galactosidases towards 2-amino benzoic acid labeled di- and trisaccharides (lactose-AA: Galβ1,4Glc-AA; galacto-N-biose-AA: Galβ1,3GalNAc-AA; 2-fucosyllactose-AA: Fucα1,2Galβ1,4Glc-AA; 3-fucosyllactose-AA: Fucα1,3[Galβ1,4]Glc-AA; N-acetyllactosamine-AA: Galβ1,4GlcNAc-AA and Galβ1,6GlcNAc-AA) were analyzed on reverse-phase HPLC (ODS HypersilTM, 250 × 4 mm, ThermoFisher Scientific–Bonn, Germany) with solvent A: 0.2% (v/v) 1-butylamin, 0.5% (v/v) orthophosphoric acid, 1% (v/v) tetrahydrofuran in H₂O and solvent B: solvent A/acetonitrile = 50/50 (v/v). The elution was performed by a linear gradient of solvent B from 5–100% in 23 min, at a flow rate of 1 mL/min. Quantification was done by peak integration after fluorescence detection at ex/em 360 nm/425 nm [33 (link)].
Separation of pNP-labeled sugars (pNP-lactose: pNP-Glcβ1,4Gal, pNP-galacto-N-biose: pNP-GalNAcβ1,3Gal) was done on reverse-phase HPLC (ODS HypersilTM, 250 × 4.6 mm, ThermoFisher Scientific–Bonn, Germany) with solvent A composing of 0.1 M ammonium acetate, pH 6.0 and solvent B containing 50% (v/v) acetonitrile in H₂O. Elution was achieved by a linear gradient of solvent B from 5–50% in 30 min, at a flow rate of 1 mL/min. Quantitative values were obtained by peak integration after UV detection at 280 nm.

Cysteine (Cys), cysteinyl-glycine (Cys-Gly), homocysteine (Hcy), and glutathione (GSH) were analyzed as previously reported [16 (link)]. We updated the chromatographic system, thus the high-performance liquid chromatography (HPLC) was an Agilent Technologies 1290 Infinity System (Agilent Technologies, Waldbronn, Germany, EU) furnished with a fluorescence detector operating at an excitation wavelength of 390 nm and an emission wavelength of 478 nm. The signals acquired were examined by the Agilent OpenLab CDS ChemStation Edition (software vA.02.15; Agilent Technologies, Waldbronn, Germany, EU). Column (Hypersil ODS; 150 × 4.6 mm, 3 μm particle size) and precolumn (Hypersil ODS; 10 × 4 mm, 5 μm particle size) were supplied by Thermo Scientific (Thermo Fisher Scientific, Bellefonte, PA, USA).

Extraction of rebeccamycin from 20 mL cultivation broth was performed using 5 mL ethyl acetate which was incubated for 60 min in an overhead shaker (Intelli-Mixer RM-2 M, LTF Labortechnik, Wasserburg, Germany). After centrifugation for 10 min at 4000 min^-1 (Heraeus Varifuge 3.0R, Thermo Fisher Scientific, Waltham, USA) the ethyl acetate was removed and used for HPLC measurements (HitachiLaChrom Elite, Hitachi, Tokyo, Japan). For HPLC analysis a pre-column Hypersil ODS (5 μm, 50 × 4.6 mm, Thermo Fisher Scientific, Waltham, USA) and a column Hypersil ODS (5 μm, 250 × 4.6 mm, Thermo Fisher Scientific, Waltham, USA) at 30°C were used. A gradient of two eluents, 0.1% trifluoric acid (eluent A) and 90% acetonitrile (eluent B), was used at a flow rate of 1 mL min^-1. Starting at 77.8% of eluent A, it was reduced to 16.6% eluent A over 20 min, kept constant for 2 min and was increased to 77.8% again. A diode array detector (Hitachi L-2455 Diode Array Detector, Hitachi, Tokyo, Japan) detected rebeccamycin at a wavelength of 316 nm with a retention time of approximately 18 min.
The biomass-specific rebeccamycin productivity is calculated by
$q_P\left[\mathrm{m}\mathrm{g}{\mathrm{g}}^{-1}{\mathrm{d}}^{-1}\right]=\frac{1}{X}\bullet \frac{dP}{dt}$
with the cell dry weight concentration X and the rebeccamycin concentration P.