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Development of an Approach to Determining Enzymatic Activity of Ribonucleoside Hydrolase C Using Hydrophilic Interaction Liquid Chromatographyстатья Исследовательская статья
Статья опубликована в высокорейтинговом журнале
Информация о цитировании статьи получена из
Scopus
Статья опубликована в журнале из списка Web of Science и/или Scopus
Дата последнего поиска статьи во внешних источниках: 15 февраля 2024 г.
- Авторы: Shaposhnikov L.A., Chikurova N.Yu, Chernobrovkina A.V., Tishkov V.I., Pometun A.A.
- Журнал: Journal of Chromatography A
- Том: 1715
- Год издания: 2024
- Издательство: Elsevier BV
- Местоположение издательства: Netherlands
- Номер статьи: 464561
- DOI: 10.1016/j.chroma.2023.464561
- Аннотация: Ribonucleoside hydrolase C (RihC, EC 3.2.2.1-3.2.2.3, 3.2.2.7, 3.2.2.8) belongs to the family of ribonucleoside hydrolases that catalyze the cleavage of both purine and pyrimidine ribonucleosides to nitrogenous bases and ribose. Its most efficient reaction is the cleavage of uridine with the highest reaction rate. The reaction cannot be detected by a simple spectrophotometric method because of the same absorption maximum for the substrate and reaction product or requires time- and labor-consuming sample preparation for ribose. Reversed-phase HPLC is currently used to register enzymatic activity, where the time of one chromatographic run takes about 10 min. Since a large number of analyses are required to measure the kinetics of an enzymatic reaction, the total time is significant. In this work, we obtained new recombinant RihC from Limosilactobacillus reuteri by gene cloning and expression in E.coli cells. We proposed a new approach for determining the enzymatic activity of the new RihC using hydrophilic interaction liquid chromatography (HILIC). The novel column was developed for this procedure providing the determination of uracil and uridine with high efficiency and retention times of 0.9 and 1.7 min, respectively. Kinetic parameters for RihC uridine cleavage were determined. The proposed approach provided significant rapidity for measurement of the enzyme kinetics being 5 times faster as compared to reversed-phase HPLC.
- Добавил в систему: Чернобровкина Алла Валерьевна
Прикрепленные файлы
| № | Имя | Описание | Имя файла | Размер | Добавлен |
|---|---|---|---|---|---|
| 1. | Экспертное заключение | Ekspertnoe_zaklyuchenie_JCA.PDF | 1,2 МБ | 13 мая 2024 [Natalia_Chikurova] | |
| 2. | Полный текст | 1-s2.0-S0021967323007860-main.pdf | 830,0 КБ | 2 января 2024 [Shaposhnikov_leo] |