一种在微孔板系统中使用4-甲基伞形酮标记的荧光底物测量单个菌根根尖中酶活性的快速且高度灵敏的方法。

A rapid and highly sensitive method for measuring enzyme activities in single mycorrhizal tips using 4-methylumbelliferone-labelled fluorogenic substrates in a microplate system.

作者信息

Pritsch K, Raidl S, Marksteiner E, Blaschke H, Agerer R, Schloter M, Hartmann A

机构信息

Chair of Soil Ecology, Technische Universität Munich (TUM), D-85758 Oberschleissheim, Germany.

出版信息

J Microbiol Methods. 2004 Aug;58(2):233-41. doi: 10.1016/j.mimet.2004.04.001.

DOI:10.1016/j.mimet.2004.04.001

PMID:15234521

Abstract

A microplate fluorimetric assay was developed for measuring potential activities of extracellular enzymes of individual ectomycorrhizal (EM) roots using methylumbelliferone (MU)-labelled fluorescent substrate analogues and microsieves to minimise damage due to manipulation of excised mycorrhizal roots. Control experiments revealed that enzyme activities remained stable over the whole time of the experiment suggesting a strong affinity of the studied enzymes to the fungal cell walls. The same mycorrhizal tips thus could be used repeatedly for enzyme detection and subsequently analysed for the projection area by automated image analysis. The developed system was evaluated on four different EM species measuring pH optimum and substrate saturation of phosphatase, chitinase and beta-glucosidase. The four EM species studied were Lactarius subdulcis, Russula ochroleuca, Cortinarius obtusus and Xerocomus cf. chrysenteron. Depending upon the enzyme, each species exhibited different levels of enzymatic activities as well as enzyme kinetics and showed also differences in pH optima.

摘要

开发了一种微孔板荧光测定法，用于使用甲基伞形酮（MU）标记的荧光底物类似物和微筛来测量单个外生菌根（EM）根的细胞外酶的潜在活性，以尽量减少因处理离体菌根根而造成的损害。对照实验表明，在整个实验过程中酶活性保持稳定，这表明所研究的酶对真菌细胞壁具有很强的亲和力。因此，相同的菌根尖端可重复用于酶检测，随后通过自动图像分析对投影面积进行分析。所开发的系统在四种不同的EM物种上进行了评估，测量了磷酸酶、几丁质酶和β-葡萄糖苷酶的最适pH值和底物饱和度。所研究的四种EM物种分别是淡红乳菇、赭黄红菇、钝丝膜菌和近似金红绒盖牛肝菌。根据酶的不同，每个物种表现出不同水平的酶活性以及酶动力学，并且在最适pH值上也存在差异。