Disentangling two and three domain laccases in soils: contribution of fungi, bacteria and abiotic processes to oxidative activities

Oxidative biocatalytic activities are crucial for soil organic matter (SOM) decomposition and turnover. Laccases are widespread oxidative enzymes, catalyzing the oxidation of phenolic compounds and occurring as three-domain (3D) and two-domain (2D) proteins. The 2D laccases are produced only by bacteria and catalyze polymerization reactions at alkaline pH, resulting in sequestration of organic carbon in soil. The 3D laccases are produced by fungi and bacteria and participate both in decomposition and polymerization of SOM in acidic environment. The approaches to differentiate the 2D and 3D laccase activities in situ in soil are absent. Based on the ability of 3D and 2D laccases to oxidize 2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) and on their specific sensitivities to NaN3 inhibition, we developed a new method to disentangle the 3D and 2D laccase-like oxidative activities in soils. The 3D and 2D laccases were ubiquitous in Retisols, Luvisols and Chernozem soils, but their activities were dependent on soil type and land use. The average laccase activity was 0.17 U g−1, representing 19 % of the total oxidizing activity, peaking up to 95 % (1.2 U g−1) in the litter horizon of Albic Luvisol and decreasing with soil depth to 0.01–0.37 U g−1. Laccase activity in organic horizons of forest soils was three times larger than in grasslands, reflecting the importance of tree litter inputs for biocatalytic carbon turnover. Soil moisture and organic carbon contents were the main factors controlling laccase-like oxidation activities. High non-laccase (residual) activities of 0.13–2.6 U g−1 (ca. 80 % of total oxidation) were closely correlated with Mn content suggesting the contribution of abiotic factors to ABTS oxidation. This is the first report on differential analysis of 2D and 3D laccase activities in soils – an important step to understand and disentangle various biotic and abiotic drivers of SOM turnover. © 2025 Elsevier B.V., All rights reserved.