Optimization for the determination of phenol oxidase activity in subtropical forest soils developed on sandstone

Abstract

Phenol oxidase plays an important role in the degradation of soil organic matter. There was no standard method to determine soil phenol oxidase activity. To fill such knowledge gap, we investigated the effects of substrate type, pH, soil storage conditions, storage time, substrate concentration, water-soil ratio, incubation time and incubation temperature on soil phenol oxidase activity in three different subtropical forest soils developed on sandstone. The pH of extraction buffers significantly affected the phenol oxidase activity. Using 2,2'-azinobis-(-3-ethylbenzo-thiazoline-6-sulfononic acid)-diammonium salt (ABTS) as substrate acquired higher oxidase activity and was applicable to wider pH range than using 3-(3,4-Dihydroxyphenyl)-L-alanine (L-DOPA) as substrate, indicating that ABTS was more suitable as a substrate for measuring phenol oxidase activity in acidic soils of subtropical forests. The storage condition significantly affected phenol oxidase activity. The phenol oxidase activity declined with time in all the three types of soil. The decreasing rate was air-dried > 4 ℃ refrigerated > -20 ℃ frozen > -80 ℃ frozen, suggesting that the frozen storage method was better than others in maintaining soil phenol oxidase activity if the determination of phenol oxidase activity in fresh soil samples cannot be immediately done. Substrate concentration, water-soil ratio, and incubation time and temperature all affected the activity of soil phenol oxidase. The condition of soil: buffer ratio of 1:100, 2 mmol·L-1 concentration of ABTS with an incubation time of 4 h at 25-30 ℃ was optimal for measuring phenol oxidase activity in acidic soils of subtropical forests, with high repeatability and sensitivity.