Stability of the fluorogenic enzyme substrates and pH optima of enzyme activities in different Finnish soils

Fluorogenic artificial substrates facilitate sensitive enzyme activity measurements for a variety of processes in soil and other environmental samples. It is possible to use in situ pH for measurements on condition that the substrates are chemically stable. We studied the stability of 12 different methyl umbellipherone (MUF) and amino methyl coumarine (AMC) derivatives used as substrates for arylsulphatase, alpha-glucosidase, beta-glucosidase, beta-xylosidase, cellobiosidase, chitinase, phosphomonoesterase (PME), phoshodiesterase (PDE), esterase, lipase and alanine- and leucine aminopeptidases (AP) over the pH range from 4.0 to 8.0 in modified universal buffer (MUB). Stability of the substrates for lipase (4-MUF-heptanoate) and esterase (4-MUF-acetate) measurements was poor, especially at the higher pH values. Chitinase substrate, 4-MUF-N-acetyl-beta-D-glucosamide, was unstable at high pH values whereas the substrate for PME activity measurement (4-MUF-phosphate) disintegrated at low pH. The other substrates and MUF and AMC standard solutions were stable over the pH range studied. The optima between pH 4 and 8 of the 11 different enzyme activities were measured in three forest and two agricultural soil samples and in one activated sludge sample. In soil, for alanine and leucine AP the pH optima were usually 7.5 or higher, for arylsulphatase, beta-glucosidase, beta-xylosidase, esterase and PDE between 4 and 5.5, and for cellobiosidase between 4 and 5. alpha-Glucosidase had an optimum below 5.5 but also exhibited high activity at pH 7. Soil-dependent variation in pH optima were observed for chitinase, esterase, PDE and PME. Enzyme activities were also measured in 0.5 M acetate buffer at pH 5.5. This buffer yielded the highest activities in all soil samples for arylsulphatase, PDE and PME.