Evaluation of microplate and bench-scale β-glucosidase assays for reproducibility, comparability, kinetics, and homogenization methods in two soils

Abstract

Enzyme assays that use fluorescently labeled substrates and microplate formats have been incorporated into laboratory protocols to improve sensitivity and reduce the time and labor involved in traditional bench-scale analyses. Microplate protocols vary, and the methods have not been evaluated systematically for comparability and reproducibility. In this study, p-nitrophenol (pNP)-based and 4-methylumbelliferone (MUF)-based microplate methods for estimating β-glucosidase activity were compared in two soils with different properties. Microplate method reproducibility was evaluated in replicate soil suspensions, and Michaelis–Menten kinetics for the microplate assays were compared to those of a standard pNP bench-scale assay. The effect of soil sample sonication on reproducibility was determined for the MUF microplate method. The MUF microplate method was reproducible in five replicate soil suspensions, but the pNP microplate method showed greater variability. The Km Michaelis–Menten constant was significantly different in the microplate methods compared to the bench method. Enzyme activities measured by the MUF and bench methods were comparable, but the pNP microplate method resulted in more variable measurements and was less sensitive in the soils studied. Sonication of soil at an intensity of 15 W ml−1 resulted in higher (MUF) measurements, but greater variability. The effects of high background absorbance on the reproducibility, sensitivity, and accuracy of the pNP microplate method do not support this method as a substitute for the standard bench method. A robust comparison study of the MUF microplate method across laboratories is recommended to further validate its use in comparative analyses.