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An automated method to evaluate the enzyme kinetics ofglucosidases
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SYSNO ASEP 0506682 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title An automated method to evaluate the enzyme kinetics ofglucosidases Author(s) Klimeš, P. (CZ)
Mazura, P. (CZ)
Turek, D. (CZ)
Brzobohatý, Břetislav (BFU-R) RID, ORCIDNumber of authors 4 Source Title Protein Science. - : Wiley - ISSN 0961-8368
Roč. 26, č. 2 (2017), s. 382-388Number of pages 7 s. Publication form Print - P Language eng - English Country US - United States Keywords maize beta-glucosidase ; cytokinin ; zm-p60.1 ; peroxidase Subject RIV CE - Biochemistry OECD category Biochemistry and molecular biology Method of publishing Open access Institutional support BFU-R - RVO:68081707 UT WOS 000393960300021 DOI 10.1002/pro.3078 Annotation Enzyme kinetic measurements are important for the characterization and engineering of biocatalysts, with applications in a wide range of research fields. The measurement of initial reaction velocity is usually slow and laborious, which motivated us to explore the possibilities for automating this process. Our model enzyme is the maizeglucosidase Zm-p60.1. Zm-p60.1 plays a significant role in plant growth and development by regulating levels of the active plant hormone cytokinin. Zm-p60.1 belongs to a wide group of hydrolytic enzymes. Members of this group hydrolyze several different types of glucosides, releasing glucose as a secondary product. Enzyme kinetic measurements using artificial substrates are well established, but burdensome and time-consuming. Thus, they are a suitable target for process automation. Simple optical methods for enzyme kinetic measurements using natural substrates are often impossible given the optical properties of the enzymatic reaction products. However, we have developed an automated method based on glucose detection, as glucose is released from all substrates of glucosidase reactions. The presented method can obtain 24 data points from up to 15 substrate concentrations to precisely describe the enzyme kinetics. The combination of an automated liquid handling process with assays that have been optimized for measuring the initial hydrolysis velocity ofglucosidases yields two distinct methods that are faster, cheaper, and more accurate than the established protocols. Workplace Institute of Biophysics Contact Jana Poláková, polakova@ibp.cz, Tel.: 541 517 244 Year of Publishing 2020 Electronic address https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/pro.3078
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