esoilБюллетень Почвенного института имени В.В. ДокучаеваDokuchaev Soil Bulletin0136-16942312-4202V.V. Dokuchaev Soil Science Institute10.19047/0136-1694-2022-113-58-89esoil-732Research ArticleСтатьиЗимография: особенности постановки метода визуализации активности ферментов в почвахZymography: developing of the enzyme soil activity visualization methodТимофееваМ. В.TimofeevaM. V.

Закончила магистратуру кафедры общего почвоведения (факультет почвоведения МГУ) в 2019.

Область интересов: функционирование криогенных почв, торфяные почвы, методические особенности определения параметров биологической активности почв 

mtimofeeva02@gmail.comhttps://orcid.org/0000-0002-3552-8054АбросимовК. Н.AbrosimovK. N.Abrosimov_KN@esoil.ruhttps://orcid.org/0000-0003-2453-3090ЮдинаА. В.YudinaA. V.anna.v.yudina@gmail.comhttps://orcid.org/0000-0003-3733-0284ФоминД. С.FominD. S.dsfomin92@gmail.comhttps://orcid.org/0000-0003-4823-444XКлюеваВ. В.KlyuevaV. V.vvklyueva@gmail.comФГБНУ ФИЦ "Почвенный институт имени В.В.Докучаева"РоссияFederal Research Centre “V.V. Dokuchaev Soil Science Institute”Russian Federation20223012202201135889Copyright © Тимофеева М.В., Абросимов К.Н., Юдина А.В., Фомин Д.С., Клюева В.В., 20232023Тимофеева М.В., Абросимов К.Н., Юдина А.В., Фомин Д.С., Клюева В.В.Timofeeva M.V., Abrosimov K.N., Yudina A.V., Fomin D.S., Klyueva V.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://bulletin.esoil.ru/jour/article/view/732

Ферменты, продуцируемые почвенной биотой, являются ключевым звеном в регуляции биохимических процессов. Активность ферментов в почве может быть визуализирована с помощью зимографии – метода, основанного на применении флуоресцирующих субстратов и позволяющего получать двумерные изображения (зимограммы). Авторами предложен вариант зимографической установки, представлены параметры освещения, фотоаппаратуры и режимов съемки, подготовка реактивов и калибровка. На примере контрастных по гранулометрическому составу почв (песок и тяжелый суглинок) детально описаны процессы подготовки образцов к анализу и сам анализ. Рассмотрены различные варианты внесения субстрата: раскапывание, окунание, насыщение, также проведена оценка влияния длительности инкубирования образцов на качество получаемых изображений. Оценены возможности и недостатки метода, предложены варианты решения методических проблем, возникающих в процессе анализа. Зимография – перспективный метод, позволяющий сопоставить данные с результатами других методов. Использование нейросетевых технологий позволяет с высокой достоверностью получить объемное распределение почвенных ферментов. Зимография требует качественно проделанной подготовительной работы и предельной аккуратности при проведении анализа. Необходимо обеспечить максимальный контакт между субстратом и почвой, так как это один из ключевых факторов, определяющих качество результатов. Установлено, что наиболее оптимальным способом внесения субстрата является насыщение мембран в растворе на протяжении 60 минут. На данном этапе развития метода нет возможности установить универсальное время инкубирования образцов, так как оно зависит от ряда характеристик как исследуемой почвы, так и условий проведения зимографии. При презентации результатов метода необходимо детально документировать условия его проведения.

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.

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The authors declare that there are no conflicts of interest present.