亚热带砂岩发育森林土壤酚氧化酶活性测定方法优化

酚氧化酶在土壤有机质降解过程中起重要作用,然而,目前用于测定土壤酚氧化酶活性的方法尚未统一。本研究以亚热带地区砂岩发育的3种不同林分的森林土壤为对象,探讨底物类型、pH值、土壤储存条件、储存时间、底物浓度、水土比、培养时间和温度对土壤酚氧化酶活性的影响,以期建立统一、可比较的测定亚热带森林土壤酚氧化酶活性的方法。结果表明: 浸提液pH值显著影响土壤酚氧化酶活性,且与目前普遍使用的左旋多巴胺(L-DOPA)相比,2,2′-联氨-双(3-乙基苯并噻唑啉-6-磺酸)-二胺盐(ABTS)所测得的氧化酶活性更高、适用pH值范围更广,说明ABTS可能更适合作为测定亚热带森林酸性土壤酚氧化酶活性的底物。储存方式显著影响酚氧化酶活性,3种供试土壤样品酚氧化酶活性均随时间呈降低的趋势,降幅表现为风干> 4 ℃冷藏> -20 ℃冷冻> -80 ℃冷冻,表明在无法保证快速测定土壤酚氧化酶活性的情况下,冷冻保存方式更有利于维持土壤酚氧化酶活性。底物浓度、水土比以及培养时间和温度均影响土壤酚氧化酶活性。当土壤样品与浸提液比例为1∶100时,选择2 mmol·L^-1浓度的ABTS为底物,在25~30 ℃下培养4 h,测定酚氧化酶活性结果重复性好、灵敏度高,是测定亚热带森林酸性土壤酚氧化酶活性的最优条件。

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

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.