An automated method to evaluate the enzyme kinetics ofglucosidases

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F68081707%3A_____%2F17%3A00506682" target="_blank" >RIV/68081707:_____/17:00506682 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/62156489:43210/17:43912527

Výsledek na webu

<a href="https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/pro.3078" target="_blank" >https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/pro.3078</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1002/pro.3078" target="_blank" >10.1002/pro.3078</a>

Jazyk výsledku

angličtina

Název v původním jazyce

An automated method to evaluate the enzyme kinetics ofglucosidases

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

An automated method to evaluate the enzyme kinetics ofglucosidases

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

—

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2017

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Protein Science

ISSN

0961-8368

e-ISSN

—

Svazek periodika

26

Číslo periodika v rámci svazku

2

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

7

Strana od-do

382-388

Kód UT WoS článku

000393960300021

EID výsledku v databázi Scopus

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