Enzyme Assays for Lignocellulose Degradation

For lignin-degrading enzymes, MnP activity was measured by monitoring the oxidation of 1-mM MnSO₄ (ε₂₇₀ = 11,590/M/cm) at 270 nm, in a buffer containing 50-mM pH 5.0 malonate, 1-mM MnSO₄, and 0.1-mM H₂O₂. One unit of MnP activity was defined as the amount of enzyme that oxidized 1 μmol of Mn²⁺ per min at 25 °C [52 (link)]. The Mn²⁺-independent activity was measured by monitoring the oxidation of 1-mM ABTS (ε₄₂₀ = 36,000/M/cm) at 420 nm, in a buffer containing 50-mM pH 5.0 malonate and 0.1-mM H₂O₂. One unit of Mn²⁺-independent activity was defined as the amount of enzyme that oxidized 1 μmol of ABTS per min at 25 °C [55 (link)]. Iron-reducing activity was determined by forming Fe²⁺–ferrozine complex in the 50-mM pH 4.8 acetate buffer containing 0.3-mM FeCl₃ and 4-mM ferrozine. One unit of iron-reducing activity was defined as the rate of absorbance increase at 562 nm/min. The CDH activity was determined by oxidation of 0.3-mM 2,6-dichloroindophenol sodium salt (DCPIP, ε₅₂₀ = 68,000/M/cm) at 520 nm in the presence of 30-mM lactose in the 50-mM pH 4.8 acetate buffer. One unit of CDH activity was defined as the amount of enzyme that oxidized 1 μmol of DCPIP per min at 25 °C [76 (link)]. For cellulose- and hemicellulose-degrading enzymes, the overall cellulase activity, EG, BG, and xylanase activities were determined according to the method described by Xu et al. [63 (link)]. The esterase activity was determined using 1-mM p-nitrophenyl butyrate (ε₃₄₈ = 8321/M/cm) at 348 nm in the 50-mM pH 6.0 sodium phosphate buffer. One unit of esterase activity was defined as the amount of enzyme that released 1 μmol of p-nitrophenol per min at 25 °C [77 (link)].