Exploration of Enzyme Diversity by Integrating Bioinformatics with Expression Analysis and Biochemical Characterization

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14310%2F18%3A00101756" target="_blank" >RIV/00216224:14310/18:00101756 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00159816:_____/18:00068622

Výsledek na webu

<a href="http://dx.doi.org/10.1021/acscatal.7b03523" target="_blank" >http://dx.doi.org/10.1021/acscatal.7b03523</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1021/acscatal.7b03523" target="_blank" >10.1021/acscatal.7b03523</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Exploration of Enzyme Diversity by Integrating Bioinformatics with Expression Analysis and Biochemical Characterization

Popis výsledku v původním jazyce

Millions of protein sequences are being discovered at an incredible pace, representing an inexhaustible source of biocatalysts. Here, we describe an integrated system for automated in silico screening and systematic characterization of diverse family members. The workflow consists of (i) identification and computational characterization of relevant genes by sequence/structural bioinformatics, (ii) expression analysis and activity screening of selected proteins, and (iii) complete biochemical/biophysical characterization and was validated against the haloalkane dehalogenase family. The sequence-based search identified 658 potential dehalogenases. The subsequent structural bioinformatics prioritized and selected 20 candidates for exploration of protein functional diversity. Out of these 20, the expression analysis and the robotic screening of enzymatic activity provided 8 soluble proteins with dehalogenase activity. The enzymes discovered originated from genetically unrelated Bacteria, Eukaryota, and also Archaea. Overall, the integrated system provided biocatalysts with broad catalytic diversity showing unique substrate specificity profiles, covering a wide range of optimal operational temperature from 20 to 70 degrees C and an unusually broad pH range from 5.7 to 10. We obtained the most catalytically proficient native haloalkane dehalogenase enzyme to date (k(cat)/K-0.5 = 96.8 mM(-1) s(-1) the most thermostable enzyme with melting temperature 71 degrees C, three different cold-adapted enzymes showing dehalogenase activity at near-to-zero temperatures, and a biocatalyst degrading the warfare chemical sulfur mustard. The established strategy can be adapted to other enzyme families for exploration of their biocatalytic diversity in a large sequence space continuously growing due to the use of next-generation sequencing technologies.

Název v anglickém jazyce

Exploration of Enzyme Diversity by Integrating Bioinformatics with Expression Analysis and Biochemical Characterization

Popis výsledku anglicky

Millions of protein sequences are being discovered at an incredible pace, representing an inexhaustible source of biocatalysts. Here, we describe an integrated system for automated in silico screening and systematic characterization of diverse family members. The workflow consists of (i) identification and computational characterization of relevant genes by sequence/structural bioinformatics, (ii) expression analysis and activity screening of selected proteins, and (iii) complete biochemical/biophysical characterization and was validated against the haloalkane dehalogenase family. The sequence-based search identified 658 potential dehalogenases. The subsequent structural bioinformatics prioritized and selected 20 candidates for exploration of protein functional diversity. Out of these 20, the expression analysis and the robotic screening of enzymatic activity provided 8 soluble proteins with dehalogenase activity. The enzymes discovered originated from genetically unrelated Bacteria, Eukaryota, and also Archaea. Overall, the integrated system provided biocatalysts with broad catalytic diversity showing unique substrate specificity profiles, covering a wide range of optimal operational temperature from 20 to 70 degrees C and an unusually broad pH range from 5.7 to 10. We obtained the most catalytically proficient native haloalkane dehalogenase enzyme to date (k(cat)/K-0.5 = 96.8 mM(-1) s(-1) the most thermostable enzyme with melting temperature 71 degrees C, three different cold-adapted enzymes showing dehalogenase activity at near-to-zero temperatures, and a biocatalyst degrading the warfare chemical sulfur mustard. The established strategy can be adapted to other enzyme families for exploration of their biocatalytic diversity in a large sequence space continuously growing due to the use of next-generation sequencing technologies.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

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)

Rok uplatnění

2018

Kód důvěrnosti údajů

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

Název periodika

ACS Catalysis

ISSN

2155-5435

e-ISSN

—

Svazek periodika

8

Číslo periodika v rámci svazku

3

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

21

Strana od-do

2402-2412

Kód UT WoS článku

000426804100087

EID výsledku v databázi Scopus

—