Exploring mechanism of enzyme catalysis by on-chip transient kinetics coupled with global data analysis and molecular modeling

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00159816%3A_____%2F21%3A00075213" target="_blank" >RIV/00159816:_____/21:00075213 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00216224:14310/21:00122285

Výsledek na webu

<a href="https://www.sciencedirect.com/science/article/abs/pii/S2451929421000917?via%3Dihub" target="_blank" >https://www.sciencedirect.com/science/article/abs/pii/S2451929421000917?via%3Dihub</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.chempr.2021.02.011" target="_blank" >10.1016/j.chempr.2021.02.011</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Exploring mechanism of enzyme catalysis by on-chip transient kinetics coupled with global data analysis and molecular modeling

Popis výsledku v původním jazyce

The ability to engineer enzymes for industrial and biomedical applications is primarily limited by a paucity of mechanistic understanding. To gain insight into the mechanisms of enzyme catalysis, one must screen enormous numbers of discrete reaction conditions, which is a laborious task using conventional technologies. To address such limitations, we develop a droplet-based microfluidic platform for high-throughput acquisition of transient kinetic data over a range of substrate concentrations and temperatures. When compared with conventional methods, our platform reduces assay volumes by six orders of magnitude and increases throughput to 9,000 reactions/min. To demonstrate their utility, we measure the transient kinetics of three model enzymes, namely, beta-galactosidase, horseradish peroxidase, and microperoxidase. Additionally, we conduct a complex kinetic and thermodynamic study of engineered variants of haloalkane dehalogenases. Datasets are globally analyzed and complemented by molecular dynamics simulations, providing new insights into the molecular basis of substrate specificity and the role of hydration-related entropy.

Název v anglickém jazyce

Exploring mechanism of enzyme catalysis by on-chip transient kinetics coupled with global data analysis and molecular modeling

Popis výsledku anglicky

The ability to engineer enzymes for industrial and biomedical applications is primarily limited by a paucity of mechanistic understanding. To gain insight into the mechanisms of enzyme catalysis, one must screen enormous numbers of discrete reaction conditions, which is a laborious task using conventional technologies. To address such limitations, we develop a droplet-based microfluidic platform for high-throughput acquisition of transient kinetic data over a range of substrate concentrations and temperatures. When compared with conventional methods, our platform reduces assay volumes by six orders of magnitude and increases throughput to 9,000 reactions/min. To demonstrate their utility, we measure the transient kinetics of three model enzymes, namely, beta-galactosidase, horseradish peroxidase, and microperoxidase. Additionally, we conduct a complex kinetic and thermodynamic study of engineered variants of haloalkane dehalogenases. Datasets are globally analyzed and complemented by molecular dynamics simulations, providing new insights into the molecular basis of substrate specificity and the role of hydration-related entropy.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

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OECD FORD obor

10400 - Chemical sciences

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

R - Projekt Ramcoveho programu EK

Rok uplatnění

2021

Kód důvěrnosti údajů

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

Název periodika

Chem

ISSN

2451-9294

e-ISSN

—

Svazek periodika

7

Číslo periodika v rámci svazku

4

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

14

Strana od-do

1066-1079

Kód UT WoS článku

000652330200020

EID výsledku v databázi Scopus

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