生态科学2025，Vol.44Issue(5)：97-108,12.DOI:10.14108/j.cnki.1008-8873.2025.05.010
土壤碳氮磷转化关键水解酶动力学特征测定方法优化
Methods optimization of kinetic characteristics of key hydrolytic enzymes involved in soil carbon,nitrogen and phosphorus transformation
摘要
Abstract
The key enzymes involved in the conversion of soil carbon,nitrogen,and phosphorus participate in the transformation of polysaccharides,cellulose,chitin,protein,and phospholipids,which are important evaluation indicators of soil quality.Enzyme kinetic characteristics can reflect the enzymatic reaction process under different substrate concentrations more comprehensively than enzyme activity.However,there are few studies optimize soil enzyme kinetic methods with different soil physical and chemical properties.Moreover,there are many types of enzyme kinetics statistics.Microplate fluorimetry assay method was used to measure enzyme kinetic characteristics of Cinnamon soil with different physical and chemical properties.The measurement conditions were optimized as follows.Namely,the substrate concentration range,incubation time and whether to add NaOH solution as a stop solution were optimized.Besides,the layout of fluorescent microplate was optimized,and data statistics with different research aims were integrated.The results were as follows.(1)In the range of 0.96-7.67 mg·g-1 of soil total carbon content,the substrate concentration range of α-D-glucosidase,β-D-glucosidase and acid phosphatase were all 0-200 μmol·L-1,with the incubation time of 4,3,and 2.5 h,respectively.For cellobiase and L-leucine aminopeptidase,the substrate concentration range were both 0-400 μmol·L-1,with the incubation time of 4 and 2 h,respectively.For N-acetyl-β-D-glucosaminidase,the substrate concentration range was 0-800 μmol·L-1 with the incubation time of 4 h.(2)Adding NaOH stop solution is necessary in the measurement of α-D-glucosidase,cellobiase,N-acetyl-β-D-glucosaminidase.Adding NaOH stop solution can be omitted in the measurement of acid phosphatase and L-leucine aminopeptidase enzyme kinetics.(3)Statistical analysis of common soil enzyme kinetic characteristics included statistics of enzyme activities under different substrate concentrations,ecoenzymatic stoichiometry,analysis of individual soil enzyme kinetic parameters and influencing factors,and analysis of the relationship between enzyme kinetic comprehensive characteristics and influencing factors.In conclusion,the measurement conditions of kinetic characteristics of key hydrolytic enzymes involved in carbon,nitrogen and phosphorus transformation should be optimized according to soil physical and chemical properties.Furthermore,the statistical method should be chosen according to the research purpose.关键词
土壤酶动力学/微孔板荧光法/培养时间/终止液/缓冲液/统计分析Key words
soil enzyme kinetics/microplate fluorimetric assay method/incubation time/stop solution/buffer solution/statistics分类
生物学引用本文复制引用
包金美,陈法霖,张静,杨龙超,王立志,刘波,张全艳,王芸..土壤碳氮磷转化关键水解酶动力学特征测定方法优化[J].生态科学,2025,44(5):97-108,12.基金项目
国家自然科学基金项目(32071630) （32071630）
山东省自然科学基金项目(ZR2021MD045,ZR2020MD104) （ZR2021MD045,ZR2020MD104）
