Overlooked tools for studying soil nitrogen depolymerization: Aminopeptidase assays using nitroanilide substrates

Aminopeptidases are one of the extracellular hydrolytic enzymes that catalyze organic nitrogen (N) depolymerization and are commonly assayed using fluorogenic substrates. However, chromogenic substrates based on para-nitroaniline (pNA) developed for the study of aminopeptidases in the 1960s have been underutilized. To gauge the use of pNA substrates to assay soil aminopeptidases, a systematic literature review was conducted. We identified 61 studies that were nearly all limited to measuring leucine and/or glycine aminopeptidases, despite the commercial availability of at least six other aminopeptidase-specific pNA substrates. Assay parameters of scale (slurry vs. direct incubations), matrix type, buffer pH, substrate concentration, assay duration and temperature, termination, and colorimetry indicated a lack of standardization and a confusion of pNA with pNP substrates despite important differences in abiotic hydrolysis and absorbance maxima. Future studies should systematically evaluate and standardize these parameters and assess the sensitivity of other amino acid-specific aminopeptidases to carbon (C), N, and sulfur (S) cycling.