Past, Present and Future in Soil Enzymology

The bibliography on soil enzymes is extensive as showed by books and many review chapters devoted to the subject. The assays of soil enzymes are generally simple, accurate, sensitive and relatively rapid and for this reasons they have been extensively used to determine the effects of contaminants, changes in management practices and effects of environmental factors and plant cover on soil metabolism. However, the present enzyme assays determine potential rather than real enzyme activities due to the optimal conditions of the assays and they do not discriminate the contribution of extracellular stabilised enzymes from that of intracellular enzyme activities. The determination of the latter is important to evaluate the answer of soil microorganisms to any effect on soil. Methods based on fumigation of soil with chloroform or with the physiological response of soil microorganisms to glucose addition to soil present drawbacks. Presently, enzyme activities are still used to evaluate the response of soil metabolism to any effect not only in arable soils but also in forest soils. However, not always the past bibliography and the limits of the present enzyme assays are considered. A few innovative studies have been carried out. Measurements of enzyme activities have been combined with those on microbial diversity evaluated by molecular techniques. Both synthesis and persistence of phosphomonoesterases have been quantified in studies based on the stimulation of microbial growth by adding easily degradable organic compounds to soil. Metcalfe et al. (Metcalfe AC, Krsek M, Gooday GW, Prosseer JI, Wellington EM (2002) Appl Environ Microbiol 68:5042–5050) covered all events from gene presence, through gene expression and up to the detection of target enzyme in soil. The addition of sludge to a pasture soil increased chitinase activity and the number of actinobacteria but selected actinobacterium-like chitinase sequences. Enzyme assays distinguishing the contribution of extracellular stabilised enzymes from that of intracellular enzyme activities are needed. Future research should increase the number of enzyme activities which can be determined in soil. For example, an accurate assay for determining nuclease activity in soil is not available. It is important to set up accurate methods for extracting intracellular and stabilised extracellular proteins, which are largely prevailing, so as to be able to carry out the proteomic approach in soil. The understanding of microbial synthesis of proteins (functional proteomic) as affected by different environmental conditions can increase our knowledge on the synthesis of enzymes in soil whereas the characterization of proteins protected against microbial degradation by their interactions with surface-reactive particles or their inclusion within humic component (structural proteomic) can give insights on the stabilization of organic N, including enzymes, in soil. The set up of suitable techniques is needed to visualise the location of stabilised enzymes in soil sections by both scanning electron microscopy and transmission electron microscopy. Acid phosphatase activity has been detected in small (7 × 20 nm) fragments of microbial membranes, roots, mycorrhizae, etc. of soil but not in naturally-electron dense soil components (minerals) and in soil components reacting with OsO4 (humus) and this does not permit to localize extracellular enzymes or proteins stabilized by clay minerals or humic materials (Ladd JN, Butler JHA (1966) Aust J Soil Res 4:41–54).