Exploring the enzymatic landscape: distribution and kinetics of hydrolytic enzymes in soil particle-size fractions

The location of extracellular enzymes within the soil architecture and their association with the various soil components affects their catalytic potential. A soil fractionation study was carried out to investigate: (a) the distribution of a range of hydrolytic enzymes involved in C, N and P transformations, (b) the effect of the location on their respective kinetics, (c) the effect of long-term N fertilizer management on enzyme distribution and kinetic parameters. Soil (silty clay loam) from grassland which had received 0 or 200 kg N ha⁻¹ yr⁻¹ was fractionated, and four particle-size fractions (>200, 200-63, 63-2 and 0.1-2 μm) were obtained by a combination of wet-sieving and centrifugation, after low-energy ultrasonication. All fractions were assayed for four carbohydrases (β-cellobiohydrolase, N-acetyl-β-glucosaminidase, β-glucosidase and β-xylosidase), acid phosphatase and leucine-aminopeptidase using a microplate fluorimetric assay based on MUB-substrates. Enzyme kinetics (V_max and K_m) were estimated in three particle-size fractions and the unfractionated soil. The results showed that not all particle-size fractions were equally enzymatically active and that the distribution of enzymes between fractions depended on the enzyme. Carbohydrases predominated in the coarser fractions while phosphatase and leucine-aminopeptidase were predominant in the clay-size fraction. The Michaelis constant (K_m) varied among fractions, indicating that the association of the same enzyme with different particle-size fractions affected its substrate affinity. The same values of K_m were found in the same fractions from the soil under two contrasting fertilizer management regimes, indicating that the Michaelis constant was unaffected by soil changes caused by N fertilizer management.