Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

Haseong Kim; Kil Koang Kwon; Wonjae Seong; Seung-Goo Lee

doi:10.3791/54059

Multi-enzyme préalable au moyen d'un système de criblage enzymatique génétique à haut débit

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Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

Multi-enzyme préalable au moyen d'un système de criblage enzymatique génétique à haut débit

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08:10 min

August 8, 2016

DOI: 10.3791/54059-v

Haseong Kim¹, Kil Koang Kwon¹, Wonjae Seong¹, Seung-Goo Lee^1,2

¹Synthetic Biology & Bioengineering Research Center,Korea Research Institute of Bioscience and Biotechnology, ²Biosystems and Bioengineering Program,University of Science and Technology, Daejeon, South Korea

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Overview

This study introduces a high-throughput screening method utilizing a universal genetic enzyme screening system capable of assessing over 200 enzymes. The system identifies various enzymes, including lipase, cellulase, and alkaline phosphatase, by altering the substrate used.

Key Study Components

Area of Science

Enzyme engineering
Microbial biotechnology
Metagenomic screening

Background

High-throughput screening is essential for evaluating enzyme activity.
Traditional methods often require separate systems for different enzymes.
This study aims to streamline the screening process.
Utilizing a single system can enhance efficiency in enzyme research.

Purpose of Study

To develop a method for screening multiple enzymes simultaneously.
To facilitate research in enzyme engineering and metagenomics.
To provide a more efficient alternative to conventional screening techniques.

Methods Used

Development of a genetic enzyme screening system.
Utilization of a phenylene compound and GFP reporter for detection.
Screening of enzymes by changing the substrate provided.
Measurement of fluorescence to assess enzyme activity.

Main Results

Successful identification of lipase, cellulase, and alkaline phosphatase.
Demonstrated the versatility of the screening system.
Showed that the method can be applied to various enzymes.
Validated the effectiveness of using a single system for multiple enzymes.

Conclusions

The developed method significantly improves enzyme screening efficiency.
This approach can advance research in enzyme engineering.
Future applications may include broader metagenomic studies.

Frequently Asked Questions

What is the main advantage of this screening method?

The main advantage is the ability to screen multiple enzymes using a single system, enhancing efficiency.

How does the genetic enzyme screening system work?

It uses a phenylene compound that activates a GFP reporter upon enzyme activity, allowing for easy detection.

Which enzymes were identified in this study?

The study identified lipase, cellulase, and alkaline phosphatase.

What substrates were used in the screening?

The substrates included p-nitrophenyl acetate, p-nitrophenyl-β-D-cellobioside, and phenyl phosphate.

Who demonstrated the procedure?

Kil Koang Kwon, a graduate student in the laboratory, demonstrated the procedure.

Ce travail présente une méthode de criblage à haut débit utilisant un système universel de criblage enzymatique génétique qui peut théoriquement être appliqué à plus de 200 enzymes. Ici, le système de criblage unique identifie trois enzymes différentes (lipase, cellulase et phosphatase alcaline) en changeant simplement le substrat utilisé (acétate de p-nitrophényle, p-nitrophényl-β-D-cellobioside et phosphate de phényle).

L’objectif global de cette méthodologie est d’évaluer plusieurs enzymes à l’aide d’un seul système de criblage à haut débit. Cette méthode peut aider à répondre à des questions clés dans le domaine des micro-organismes et de la bio-ingénierie sur le criblage métagénomique et l’ingénierie enzymatique. Nous avons mis au point un système de criblage enzymatique génétique composé du composé de phénylène répondant au mutant MPL et de son rapporteur GFP en aval.

Une fois qu’un gène métagénomique montre l’activité enzymatique qui nous intéresse en réagissant avec le substrat fourni, un composé phénylène du produit de réaction active le mutant Dmp et déclenche l’expression du rapporteur GFP qui peut facilement être capturé par un compteur de fluorescence. Le principal avantage de cette technique est que, contrairement aux techniques de criblage conventionnelles, cette méthode unique est applicable au criblage de plusieurs types d’enzymes différentes en utilisant le substrat approprié. La démonstration de cette procédure sera faite par Kil Koang Kwon, un étudiant diplômé de notre laboratoire.

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