Soil Enzyme Activity Assays: Protocols and Interpretations.

Enzymes are central to soil ecosystem processes and environmental research. The challenge since the first soil enzyme (catalase) detected in 1899, has been and continues to be suitable analytical methodologies. The primary method has been activity based because extraction is difficult. However, assays, to be reproducible and comparable, must have controlled conditions that enable zero-order kinetics with optimized pH, substrate saturation, standardized incubation time and temperature, and a proper control. A misguided approach is to determine reaction rates under in situ pH and/or temperature; violating basic protocols principles that could disguise or even reverse treatment effects. Another common misinterpretation is the assumption that enzyme activity equals microbial activity. This can be true for some enzymes, notably dehydrogenase because it is an intracellular enzyme occurring only in viable cells. However, a wide array of hydrolytic enzymes involved in decomposition and nutrient cycling in soil are extracellular and no longer associated with viable cells, and have the capacity to hydrolyze complex substrates that are insoluble or too big to be transported across cell membranes. Activities of these enzymes are regulated by many factors, such as substrate availability, product feedback inhibition, and presence of promoters or inhibitors. Extracellular enzymes can be stabilized in the soil matrix but remain catalytic, as demonstrated in the 1950s-60s by Douglas McLaren with gamma radiation; where a large amount of urease activity (and others) occurred in “sterile” soil – later coined "abiontic" enzymes by John Skujiņš. Abiontic activity can be 40 to 60 % of the total activity, but is commonly not considered in data interpretation. In conclusion, the long history of soil enzyme activity methodology is still relevant and will complement the advances in molecular biology, microscopy, and analytical capability, to provide new insights into microbial ecology and address the agricultural and environmental challenges of the 21^st Century.