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 and Zbyněk PROKOP

Basic information

Original name

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

Authors

VAŠINA, Michal (203 Czech Republic, belonging to the institution), Pavel VAŇÁČEK (203 Czech Republic, belonging to the institution), Jiří DAMBORSKÝ (203 Czech Republic, guarantor, belonging to the institution) and Zbyněk PROKOP (203 Czech Republic, belonging to the institution)

Edition

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

Publisher

Elsevier Inc

Other information

Language

English

Type of outcome

Kapitola resp. kapitoly v odborné knize

Field of Study

20401 Chemical engineering

Country of publisher

Netherlands

Confidentiality degree

není předmětem státního či obchodního tajemství

Publication form

electronic version available online

References:

URL

RIV identification code

RIV/00216224:14310/20:00117531

Organization unit

Faculty of Science

ISBN

978-0-12-821149-6

DOI

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

UT WoS

000612532600004

Keywords in English

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

Abstract

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.

Links

EF17_043/0009632, research and development project

Name: CETOCOEN Excellence

LM2018121, research and development project

Name: Výzkumná infrastruktura RECETOX (Acronym: RECETOX RI)

Investor: Ministry of Education, Youth and Sports of the CR, RECETOX RI

Citovat

VAŠINA, Michal, Pavel VAŇÁČEK, Jiří DAMBORSKÝ and 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, p. 51-85. ISBN 978-0-12-821149-6. Available from: 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 and 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, p.~51-85. ISBN~978-0-12-821149-6. Available from: https://dx.doi.org/10.1016/bs.mie.2020.05.004.

Detailed Information on Publication Record