Abstract: In order to accurately determine rhizosphere soil extracellular enzyme activities, five key enzymes related to the C, N, and P circulation including β-glucosidase(βG), cellobiohydrolase(CBH), β-N-acetylglucosaminidase(NAG), leucine aminopeptidase(LAP), and alkaline phosphatase(AKP)were optimized by fluorescence method. The results showed that the ratio of water to soil should be higher than 300∶1(mL∶g)when using a highly sensitive microplate reader such as Cytation5. The optimal parameters for determining the activities of five extracellular enzymes were as follows: AKP and βG were used with ultrapure water as buffer solution, the soil was mixed with an XH-C vortex mixer(60 W)for 5 min, and incubated at 25 ℃ for 4 h; CBH was used with sodium acetate(0.2 mol/L, pH 5.8)as buffer solution, the soil was mixed with an XH-C vortex mixer(60 W)for 5 min, and incubated at 25 ℃ for 2 h; NAG was used with sodium acetate(0.2 mol/L, pH 5.8)as buffer solution in a shaker(150 r/min)for 60 min, and incubated at 25 ℃ for 3 h; LAP was used with ultrapure water as buffer solution in a shaker(150 r/min)for 60 min, and incubated at 25 ℃ for 1 h. The results of this study can provide a basis for improving the accurate determination of soil extracellular enzyme activities by fluorescence method.

Key words: fluorescence method, enzyme activity, rhizosphere soil, optimization