Zymography: developing of the enzyme soil activity visualization method

Abstract

The enzymes produced by the soil biota are a key link in the regulation of biochemical processes. The soil enzyme activity can be visualized with zymography, a method based on using fluorescent substrates and obtaining two-dimensional images (zymograms). A variant of a zymographic measuring system has been proposed. Characteristics of lighting, photographic equipment and shooting modes, reagents preparation and calibration are presented. Preparing and analyzing soil samples of different texture (sand and clay loam) and processing the study results have been described. The ways of introducing the substrate are considered in this study, namely pipetting, short-time dipping, and saturation. An analysis of the kinetics of incubation of samples was carried out. The possibilities and disadvantages of the method were also considered and options for solving possible methodological problems during the analysis were proposed. The zymography is a promising method that allows comparing data with the results of other methods. The use of neural network technologies makes it possible to obtain the volumetric distribution of soil enzymes with high reliability. The soil zymography requires qualitative preparatory work and extreme accuracy during the analysis. It is necessary to ensure maximum contact between the substrate and the soil, as this is one of the key factors determining the quality of the results. The most optimal way to introduce the substrate is to saturate the membranes with substrate solution for 60 minutes. At this stage of the development of the method, it is not possible to establish a universal sample incubation time, since this depends on characteristics of both the studied soils and the experiment conditions. Also, it is necessary to document the conditions in detail for discussion the study results.

Keywords

• β-glucosidase
• zymogram
• zymographic device
• methylumbelliferone