Exploration of enzyme diversity: High-throughput techniques for protein production and microscale biochemical characterization

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14310%2F20%3A00117531" target="_blank" >RIV/00216224:14310/20:00117531 - isvavai.cz</a>

Výsledek na webu

<a href="https://www.sciencedirect.com/science/article/pii/S0076687920302263?via%3Dihub" target="_blank" >https://www.sciencedirect.com/science/article/pii/S0076687920302263?via%3Dihub</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/bs.mie.2020.05.004" target="_blank" >10.1016/bs.mie.2020.05.004</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Exploration of enzyme diversity: High-throughput techniques for protein production and microscale biochemical characterization

Popis výsledku v původním jazyce

Enzymes are being increasingly utilized for acceleration of industrially and pharmaceutically critical chemical reactions. The strong demand for finding robust and efficient biocatalysts for these applications can be satisfied via the exploration of enzyme diversity. The first strategy is to mine the natural diversity, represented by millions of sequences available in the public genomic databases, by using computational approaches. Alternatively, metagenomic libraries can be targeted experimentally or computationally to explore the natural diversity of a specific environment. The second strategy, known as directed evolution, is to generate man-made diversity in the laboratory using gene mutagenesis and screen the constructed library of mutants. The selected hits must be experimentally characterized in both strategies, which currently represent the rate-limiting step in the process of diversity exploration. The traditional techniques used for biochemical characterization are time-demanding, cost, and sample volume ineffective, and low-throughput. Therefore, the development and implementation of high-throughput experimental methods are essential for discovering novel enzymes. This chapter describes the experimental protocols employing the combination of robust production and high-throughput microscale biochemical characterization of enzyme variants. We validated its applicability against the model enzyme family of haloalkane dehalogenases. These protocols can be adapted to other enzyme families, paving the way towards the functional characterization and quick identification of novel biocatalysts.

Název v anglickém jazyce

Exploration of enzyme diversity: High-throughput techniques for protein production and microscale biochemical characterization

Popis výsledku anglicky

Enzymes are being increasingly utilized for acceleration of industrially and pharmaceutically critical chemical reactions. The strong demand for finding robust and efficient biocatalysts for these applications can be satisfied via the exploration of enzyme diversity. The first strategy is to mine the natural diversity, represented by millions of sequences available in the public genomic databases, by using computational approaches. Alternatively, metagenomic libraries can be targeted experimentally or computationally to explore the natural diversity of a specific environment. The second strategy, known as directed evolution, is to generate man-made diversity in the laboratory using gene mutagenesis and screen the constructed library of mutants. The selected hits must be experimentally characterized in both strategies, which currently represent the rate-limiting step in the process of diversity exploration. The traditional techniques used for biochemical characterization are time-demanding, cost, and sample volume ineffective, and low-throughput. Therefore, the development and implementation of high-throughput experimental methods are essential for discovering novel enzymes. This chapter describes the experimental protocols employing the combination of robust production and high-throughput microscale biochemical characterization of enzyme variants. We validated its applicability against the model enzyme family of haloalkane dehalogenases. These protocols can be adapted to other enzyme families, paving the way towards the functional characterization and quick identification of novel biocatalysts.

Druh

C - Kapitola v odborné knize

CEP obor

—

OECD FORD obor

20401 - Chemical engineering (plants, products)

Projekt

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

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)<br>S - Specificky vyzkum na vysokych skolach<br>I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2020

Kód důvěrnosti údajů

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

Název knihy nebo sborníku

Methods in Enzymology - Enzyme Engineering and Evolution: General Methods

ISBN

9780128211496

Počet stran výsledku

35

Strana od-do

51-85

Počet stran knihy

343

Název nakladatele

Elsevier Inc

Místo vydání

Neuveden

Kód UT WoS kapitoly

000612532600004