The enzyme activities of NCS were determined in buffer (50 mM Tris-HCl, pH 7.4; 5 mM β-mercaptoethanol; 10% glycerine) containing the substrates 4, 5, and 80-mL crude enzyme. After incubation at 37 °C overnight, reactions were terminated by adding 1/2 volume of methanol. The reaction products were centrifuged at 13,400 × g for 15 min, and the supernatant was detected using HPLC analysis.
The column applied for analysis was a Hypersil GOLD™ C18 25005-254630 (4.6 × 250 mm, 5 mm) on a Shimadzu LC-2050C 3D system, with the temperature having set at 35 °C. A 10-mL sample was injected for HPLC analysis, and the detection wavelength was 282 nm. For NCS assay, mobile phases A (H2O + 0.1% formic acid) and B (acetonitrile) were run in the following gradient programs at 0.5 mL/min: 0–10 min, 5% B; 10–15 min, 5%–20% B; 15–15.1 min, 20%–50% B; 15.1–25 min, 95% B; and 25–30 min, 5% B.
The column applied for analysis was a Hypersil GOLD™ C18 25005-254630 (4.6 × 250 mm, 5 mm) on a Shimadzu LC-2050C 3D system, with the temperature having set at 35 °C. A 10-mL sample was injected for HPLC analysis, and the detection wavelength was 282 nm. For NCS assay, mobile phases A (H2O + 0.1% formic acid) and B (acetonitrile) were run in the following gradient programs at 0.5 mL/min: 0–10 min, 5% B; 10–15 min, 5%–20% B; 15–15.1 min, 20%–50% B; 15.1–25 min, 95% B; and 25–30 min, 5% B.