响应曲面分析法优化常用水解酶荧光测试法研究

Modified Fluorometric Soil Enzyme Assays as Optimized by Response Surface Methodology

土壤水解酶对森林生态系统的能量流动和养分循环具有重要作用,因此,正确测量土壤酶活性是酶学研究的基础,也有助于提高对地下生态循环的认识。所以,本实验选择常绿阔叶林与杉木针叶林这两种重要林分的土壤,基于综合实验设计和数学建模的优化方法-响应面分析法(RSM),对酸性磷酸酶(AP)、纤维素二糖水解酶(CBH)、β-葡糖苷酶(βG)、β-N-乙酰氨基葡萄糖苷酶(NAG)这4种常见的碳(C)、氮(N)、磷(P)水解酶的荧光测定方法进行优化。实验根据Box-Behnken设计原理,选取不同的搅拌时间、静置时间和培育时间,进行三因素三水平中心组合实验,通过回归方程确定了最优的组合条件。结果表明:土壤样品搅拌16.48 min,静置0 min,培育4 h后进行测定为最佳实验处理参数。本研究得到的实验方法可以提高酶活性测定的效率,而且简便易行,可为亚热带区域的森林土壤酶研究提供借鉴。

Soil hydrolytic enzymes play an important role in energy flow and nutrients cycling of forest ecosystems. Correct measurement of soil enzyme activity is the basis of enzymology research and also contributes to the understanding of below-ground ecological cycle. Evergreen broad-leaved forest and Chinese fir plantations are important forests in subtropical China. The fluorescence determination methods for C-acquiring enzyme [β-glucosidase(βG);cellobiosidase(CBH)], N-acquiring enzyme(β-1,4-N-acetylglucosaminidase, NAG) and P-acquiring enzyme activity(acid or alkaline phosphatase, AP) were optimized with response surface analysis(RSM). RSM is an optimization method based on comprehensive experimental design and mathematical modeling, which has been applied in many experimental optimization designs. Based on the results of the single-factor experiment and Box-Behnken principle, different stirring time, static time and incubation time were selected to carry out three-factor three-level center combination experiment, and the optimal combination conditions were determined by regression equation. The results showed that 16.48 min of stirring time, 0 min of static time and 4 h of incubation were the best experimental parameters for modified assays. There were no significant differences in enzyme activities after high-speed magnetic stirring or low-power ultrasonic pretreatment of soil. The modified method obtained in this study can improve the efficiency of enzyme activity determination, which can provide reference for soil enzyme research in subtropical forest.