Including intracellular β-glucosidase improves the sensitivity of soil enzyme-based mercury risk assessment

Mercury (Hg) is a highly toxic, persistent pollutant that poses significant threats to soil health and human safety. However, the distinct responses of intracellular and extracellular soil enzymes to Hg contamination across different soil types remain poorly understood. To address this gap, this study systematically compared the inhibitory effects of Hg on total, extracellular (extra-), and intracellular (intra-) β-glucosidase (BG) across six soil types, and further elucidated the inhibition kinetics and ecological toxicity to improve risk assessment. Results showed that intra-BG accounted for 71.74-85.80% of total-BG activity in uncontaminated soils, far exceeding the extracellular fraction. With increasing Hg concentration, intra-BG exhibited greater sensitivity than extra-BG, with inhibition rates ranging from 45.1 to 92.6% and 32.2-63.9%, respectively. Kinetic analysis revealed that the inhibition of BG fractions by Hg was highly dependent on soil characteristics, with total- and intra-BG being predominantly governed by competitive and non-competitive mechanisms, whereas extra-BG was primarily dominated by uncompetitive inhibition. Notably, the inhibition constants (K_ic) were consistently lower than the Michaelis constants (K_m), indicating that Hg has a stronger binding affinity than the substrate, thereby substantially suppressing enzymatic activity. Molecular docking further confirmed that Hg interacts with the enzyme's binding site, forming a locally stable conformation. Ecotoxicological assessments demonstrated that ED₁₀ (effective dose causing 10% inhibition), derived from V_max/K_m ratios (0.07-0.36 mg kg-1), was lower than values based on enzyme activity alone (0.11-1.08 mg kg-1). Furthermore, both ED₁₀ values followed the order: intra-BG ≤ total-BG < extra-BG. These findings highlight that the conventional reliance on extracellular enzymes alone may significantly underestimate the true ecological risks of soil Hg contamination. We therefore strongly advocate incorporating intra-BG as a critical biomarker in future risk assessments of heavy metal pollution in soil ecosystems.