Quantifying the activity of β-D-fucosidase in soil

Abstract

The objectives were to develop an assay method for detection and quantification, and determine kinetic parameters of β-D-fucosidase activity in soil. The optimal activities of this enzyme were at approximately pH 6.0 and 55 °C, respectively. The K_m values, based on hydrolyzing p-nitrophenyl-β-D-fucopyranoside, in five soils ranged from 2.40 to 2.89 mM, and the V_max values spanned from 206 to 1060 μmol p-nitrophenol released kg⁻¹ soil h⁻¹. At reaction temperatures from 10 to 40 °C, the mean temperature coefficient (Q₁₀) and activation energy (E_a) ranged from 1.67 to 1.90, and 44.22 to 56.39 kJ mol⁻¹, respectively. Employing a chromogenic p-nitrophenyl substrate, the developed method detects as little as 3.5 μmol p-nitrophenol released kg⁻¹ soil h⁻¹ (limit of detection or LOD), quantifies ≥ 10.7 μmol p-nitrophenol released kg⁻¹ soil h⁻¹ (limit of quantitation or LOQ), and had coefficients of variance (CV) of < 5.6%. β-D-fucosidases purified from some sources also hydrolyze β-D-galactoside, β-D-glucosides, and/or α-L-arabinosides. This method offers reproducibility and precision that are comparable with bench-scale assays, but also has the advantage of using a smaller amount of soil, reducing costs on reagents, supplies, labor, and waste management.