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

C 2020

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

VAŠINA, Michal, Pavel VAŇÁČEK, Jiří DAMBORSKÝ a Zbyněk PROKOP

Základní údaje

Originální název

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

Autoři

VAŠINA, Michal (203 Česká republika, domácí), Pavel VAŇÁČEK (203 Česká republika, domácí), Jiří DAMBORSKÝ (203 Česká republika, garant, domácí) a Zbyněk PROKOP (203 Česká republika, domácí)

Vydání

Neuveden, Methods in Enzymology - Enzyme Engineering and Evolution: General Methods, od s. 51-85, 35 s. 2020

Nakladatel

Elsevier Inc

Další údaje

Jazyk

angličtina

Typ výsledku

Kapitola resp. kapitoly v odborné knize

Obor

20401 Chemical engineering

Stát vydavatele

Nizozemské království

Utajení

není předmětem státního či obchodního tajemství

Forma vydání

elektronická verze "online"

Odkazy

URL

Kód RIV

RIV/00216224:14310/20:00117531

Organizační jednotka

Přírodovědecká fakulta

ISBN

978-0-12-821149-6

DOI

http://dx.doi.org/10.1016/bs.mie.2020.05.004

UT WoS

000612532600004

Klíčová slova anglicky

Enzyme diversity Genomic databases Metagenomics Directed evolution High-throughput protein production Microscale characterization Microfluidics

Štítky

rivok

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 15. 2. 2023 23:02, Mgr. Michaela Hylsová, Ph.D.

Anotace

V originále

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.

Návaznosti

EF17_043/0009632, projekt VaV

Název: CETOCOEN Excellence

LM2018121, projekt VaV

Název: Výzkumná infrastruktura RECETOX (Akronym: RECETOX RI)

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, RECETOX RI

Citovat

VAŠINA, Michal, Pavel VAŇÁČEK, Jiří DAMBORSKÝ a Zbyněk PROKOP. Exploration of enzyme diversity: High-throughput techniques for protein production and microscale biochemical characterization. Online. In Dan S. Tawfik. Methods in Enzymology - Enzyme Engineering and Evolution: General Methods. Neuveden: Elsevier Inc, 2020, s. 51-85. ISBN 978-0-12-821149-6. Dostupné z: https://dx.doi.org/10.1016/bs.mie.2020.05.004.

@inbook{1717557,
   author = {Vašina, Michal and Vaňáček, Pavel and Damborský, Jiří and Prokop, Zbyněk},
   address = {Neuveden},
   booktitle = {Methods in Enzymology - Enzyme Engineering and Evolution: General Methods},
   doi = {http://dx.doi.org/10.1016/bs.mie.2020.05.004},
   editor = {Dan S. Tawfik},
   keywords = {Enzyme diversity Genomic databases Metagenomics Directed evolution High-throughput protein production Microscale characterization Microfluidics},
   howpublished = {elektronická verze "online"},
   language = {eng},
   location = {Neuveden},
   isbn = {978-0-12-821149-6},
   note = {CZ.02.1.01/0.0/0.0/18_046/0015975 neuplatňovat.},
   pages = {51-85},
   publisher = {Elsevier Inc},
   title = {Exploration of enzyme diversity: High-throughput techniques for protein production and microscale biochemical characterization},
   url = {https://www.sciencedirect.com/science/article/pii/S0076687920302263?via%3Dihub},
   year = {2020}
}

TY  - CHAP
ID  - 1717557
AU  - Vašina, Michal - Vaňáček, Pavel - Damborský, Jiří - Prokop, Zbyněk
PY  - 2020
TI  - Exploration of enzyme diversity: High-throughput techniques for protein production and microscale biochemical characterization
VL  - Neuveden
PB  - Elsevier Inc
CY  - Neuveden
SN  - 9780128211496
N1  - CZ.02.1.01/0.0/0.0/18_046/0015975 neuplatňovat.
KW  - Enzyme diversity Genomic databases Metagenomics Directed evolution High-throughput protein production Microscale characterization Microfluidics
UR  - https://www.sciencedirect.com/science/article/pii/S0076687920302263?via%3Dihub
L2  - https://www.sciencedirect.com/science/article/pii/S0076687920302263?via%3Dihub
N2  - 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.
ER  -

VAŠINA, Michal, Pavel VAŇÁČEK, Jiří DAMBORSKÝ a Zbyněk PROKOP. Exploration of enzyme diversity: High-throughput techniques for protein production and microscale biochemical characterization. Online. In Dan S. Tawfik. \textit{Methods in Enzymology - Enzyme Engineering and Evolution: General Methods}. Neuveden: Elsevier Inc, 2020, s.~51-85. ISBN~978-0-12-821149-6. Dostupné z: https://dx.doi.org/10.1016/bs.mie.2020.05.004.

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