为准确测定土壤胞外酶活性,对β-葡萄糖苷酶(βG)、纤维二糖水解酶(CBH)、β-N-乙酰氨基葡萄糖苷酶(NAG)和亮氨酸氨基肽酶(LAP)、碱性磷酸酶(AKP)5种有关C、N、P循环的关键酶的荧光法测定进行了优化.结果发现,使用Cytation5等高灵敏度的酶标仪测定时水土比值应高于300∶1(mL∶g).5种胞外酶活性的最佳测定参数为:AKP、βG用超纯水作缓冲溶液,XH-C漩涡混匀器(60 W)混匀土壤5 min,25 ℃培养4 h;CBH用乙酸钠(0.2 mol/L,pH 5.8)作缓冲溶液,XH-C漩涡混匀器(60 W)混匀土壤5 min,25 ℃培养2 h;NAG用乙酸钠(0.2 mol/L,pH 5.8)作缓冲溶液,摇床(150 r/min)混匀60 min,25 ℃培养3 h;LAP用超纯水作缓冲溶液,摇床(150 r/min)混匀60 min,25 ℃培养1 h.本研究结论可为提高荧光法准确测定土壤胞外酶活性提供依据.
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.
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