Bench scale and microplate format assay of soil enzyme activities using spectroscopic and fluorometric approaches

Method standardization and validation are essential for meaningful data comparison and interpretation. The objective of this study was to compare bench scale and microplate format assays of soil enzyme activities using spectroscopic (p-nitrophenyl or pNP) and fluorometric (4-methylumbelliferyl or MUF) based approaches. Three assay approaches, including pNP-bench, pNP-microplate and MUF-microplate, were compared. Data from microplate format assays in the presence of soil suspension suggested that MUF-based assays were about 14 times more sensitive than pNP-based assays. MUF detection provided measures as low as 50 pmol of MUF in a microplate well. However, the pNP bench scale assay was the most sensitive of the three assays in quantifying enzyme activities. The limit of quantification (LOQ) values expressed as corresponding enzyme activities using the protocols tested were 0.049, 0.242, and 0.0936 mmol kg−1 h−1 for pNP bench, pNP microplate, and MUF microplate assays, respectively. Of the microplate assays, the MUF-based assay was more precise than the procedure using pNP. The presence of soil suspension increased standard errors, which more than doubled for the detection of pNP in microplate assays, but showed little effect on the standard error for the detection of MUF. For meaningful data interpretation, we suggest that LOQ values be calculated for enzyme assays and caution be exercised when interpreting data below LOQ values. Based on evaluations of three different enzymes in 16 diverse soils, the activities measured by the three protocols were often significantly different, but were in the same order of magnitude and significantly correlated, suggesting that the same pool of isoenzymes were measured by the different protocols.

► p-Nitrophenol (pNP) bench assays are more precise and sensitive than microplate based enzyme assays.
► Of microplate assays, MUF-based were about 14 times more sensitive than pNP based assays.
► The presence of soil doubled standard error on detecting pNP, but had little effect on MUF.
► Activities determined by the three protocols were comparable and significantly correlated.
► Activity below limit of quantification should be excluded or cautiously interpreted.