Assay of cellulose 1,4-β-cellobiosidase activity in soil

Abstract

As the most abundant biopolymer on earth, cellulose undergoes degradation by a diverse set of enzymes with varying specificities that act in synergism. An assay protocol was developed to detect and quantify activity of cellulose 1,4-β-cellobiosidase (EC 3.2.1.91) in soil. The optimum pH and temperature for β-cellobiosidase activity were approximately pH 5.5 and 60 °C, respectively. In the tested six soils, the Michaelis constants (K_m) ranged from 0.08 to 0.51 mM, and maximum velocity (V_max) ranged from 71.5 to 318.1 μmol kg soil^-1 h^-1. The temperature coefficient (Q₁₀) ranged from 1.72 to 1.99 at non-denaturing temperatures from 10 to 50 °C, and the activation energy (E_a) ranged from 42.5 to 53.7 kJ mol^-1. The assay procedure provided reproducible results with a coefficient of variance ≤4.7% and demonstrated a limit of quantification (LOQ) of 50.9 μmol p-nitrophenol release kg^-1 soil h^-1 for β-cellobiosidase activity in soil. Notably, the developed assay protocol offers reproducibility and precision comparable to bench-scale assays while reducing costs associated with reagents, supplies, and labor.

Keywords: CBH; Cellobiosidase; Cellulose degrading enzyme; Enzyme assay method; Enzyme kinetics; Temperature dependence.