Waters fraction collector 3

Firstly, pure ethanol was added to the R. philippinarum hydrolysates (RPHs) to achieve a concentration of 60% (v/v), and left at 4 °C for 12 h. The mixture was then centrifuged, precipitated and the supernatant was collected and freeze-dried for further use.
The fraction displaying the highest DPP-IV inhibitory activity following ethanol fractionation was further separated by RP-HPLC using a Waters 2545-2489 HPLC system (Waters Corp., Milford, MA, USA), and a Waters Xbridge Prep C₁₈ (5 μm, 19 × 150 mm) was used for separation. Elution was carried out with 2% B for 3 min, a linear gradient from 2 to 35% B for another 9 min, and kept at 35% B for 4 min, with a flow rate of 10 mL/min (eluting solvent A: 0.1% TFA in pure water; B: 0.1% TFA in methanol). The detection wavelength was set at 220 nm for all compounds. The HPLC profile is shown in Figure 2A. In total, eight fractions (M1–M8) were collected by the Waters Fraction Collector III (Waters Corp.).

Reaction mixtures containing 33 µM purified AsqJ_wt (AsqJ_V72I, AsqJ_V72K, or, AsqJ_F139I), 200 µM of the synthesized substrate^{2 (link)}, 100 µM FeSO₄, 2.5 mM α-ketoglutarate, 4 mM ascorbic acid, and 50 mM Tris hydrochloride were incubated at 30 °C for 20 s, 1 min, 2 min, 5 min, 10 min, 30 min, and 60 min. The reaction was stopped by adding 10% (v/v) of 3 M trichloroacetic acid, and samples were centrifuged at 10,000 ×  g for 10 min. All traces were monitored at 280 nm using a Dionex UltiMate 3000 HPLC system coupled with a Thermo LCQ fleet in combination with a Waters 1525 binary HPLC pump, X-Bridge Prep C18 column (5 µm, 10 × 250 mm), Waters 2998 PDA detector and Waters Fraction Collector III (Waters).

The LEGE-NPL (natural products library) solid inventory is composed of crude extracts. Thus, freeze-dried biomass was extracted three times with MeOH, with a sonication step of 5 min in between extractions, and was filtered and concentrated at 30 °C, using a rotary evaporator. The yields of extraction are described in the Supplementary Material (Figure S1). The extracts were then fractionated by reverse-phase HPLC in a Waters Alliance e2695 Separations Module instrument, coupled to a photodiode array detector (Waters 2998 PDA) and an automatic Waters Fraction Collector III (Waters, Mildford, MA, USA). Each crude was injected at 40 mg mL⁻¹ (500 µL; 1 mL loop) and separated on an ACE 10 C8 column (50 ×10 mm, ACE, Reading, UK), using a H₂O:MeCN gradient (Table 4). Hence, each cyanobacterial extract was chromatographed into eight fractions (4 mL final volume, named A–H) into 48-deep well plates (Riplate, Ritter, Schwabmünchen, Germany), which were then dried on a CentriVap Concentrator (LabConco, Kansas City, MO, USA). These fractions were solubilized in 500 µL of DMSO and transferred to 96-deep well microplates (Nest Scientific, Woodbridge Township, NJ, USA) and stored at −80 °C, thus forming the LEGE-NPL liquid library (mother plates).