2.10. Determination of Substrate Specificity of Sulfatases SWF1 and SWF4

The ability of sulfatases SWF1 and SWF4 to catalyze elimination of sulfate groups from 4-nitrophenyl sulfate, native fucoidans from brown algae F. evanescens and S. horneri, as well as sulfated fucooligosaccharides have been studied.

The effect of sulfatases on sulfated fucooligosaccharides was evaluated using C-PAGE as described above in Section 2.9.

The ability of sulfatases to catalyze the hydrolysis of sulfate groups from 4-nitrophenyl sulfate was evaluated spectrophotometrically. Reaction mixture (150 μL) containing 0.05 mg/mL sulfatase solution (SWF1 or SWF4) and 4-nitrophenyl sulfate solution 0.5 mg/mL (1.94 mM) in buffer A was icubated for 48 h at 37 °C. The reactions were stopped by adding of 100 μL of 1 M Na₂CO₃ solution, and the 4-nitrophenol produced was quantified spectrophotometrically at 410 nm using PowerWave XS plate reader (BioTek, Winooski, VT, USA).

The effect of sulfatases on native fucoidans from F. evanescens and S. horneri was studied by nuclear magnetic resonance (NMR) spectroscopy. A reaction mixture (2 mL) containing fucoidan (from F. evanescens or S. horneri, final concentration of 5 mg/mL) and 0.05 mg/mL of sulfatase (SWF1 or SWF4) solution in buffer A was dialyzed at 37 °C for 72 h against buffer A. The reaction products were deproteinised at 85 °C for 10 min and centrifuged for 10 min at 10,000× g. The supernatant was desalted using a Bio-Scale mini-column with Bio-gel P-6 (10 mL, Bio-Rad, Hercules, CA, USA) equilibrated with distilled water. The reaction products were freeze-dried and then analysed by NMR spectroscopy as described in Section 2.15.