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 (Vmax and Km) 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 (Km) varied among fractions, indicating that the association of the same enzyme with different particle-size fractions affected its substrate affinity. The same values of Km 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.