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

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

Published on: August 8, 2016

Screening assay for polyester hydrolyzing microorganisms using fluorescence-labeled poly(butylene adipate).

Bettina Semler¹, Karin Binder², Doris Ribitsch³

¹Institute of Environmental Biotechnology, Department of Agricultural Sciences, BOKU University, Vienna, Austria.

New Biotechnology

|April 7, 2025

Summary

Researchers developed a fluorescent polymer assay using 5-carboxy-fluorescein (5-FAM) coupled to poly(butylene adipate) (PBA). This method enables high-throughput screening for novel polymer-degrading enzymes and microorganisms.

Keywords:

5-carboxy-fluorescein (5-FAM)High-throughput screening Pichia pastoris Poly(butylene adipate) (PBA)Polymer-degrading enzymes

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Polymer Microarrays for High Throughput Discovery of Biomaterials

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Published on: January 25, 2012

Area of Science:

Biotechnology
Enzyme Engineering
Polymer Science

Background:

Enzymatic hydrolysis of synthetic polymers is crucial for developing sustainable materials and recycling processes.
Current methods for identifying polymer-degrading enzymes and microorganisms are often low-throughput and lack sensitivity in complex environments.

Purpose of the Study:

To develop a novel, high-throughput screening method for detecting polymer hydrolysis.
To demonstrate the utility of fluorescently labeled polymers for identifying novel enzymes and microbial sources of polymer degradation.

Main Methods:

Covalent coupling of 5-carboxy-fluorescein (5-FAM) to poly(butylene adipate) (PBA) to create fluorescently labeled polymer blends.
Incubation of labeled PBA with purified cutinase (Thc_Cut1), recombinant Pichia pastoris expressing cutinase, and fungal extracellular enzymes.
Quantification of hydrolysis by measuring fluorescence increase, released monomers, weight loss, and FTIR analysis.

Main Results:

Fluorescently labeled PBA showed a significant fluorescence increase (up to 4000 RFU) upon hydrolysis by Thc_Cut1, indicating successful detection.
The assay detected hydrolysis by recombinant Pichia pastoris (up to 500 RFU) and fungal enzymes (Fusarium solani, Alternaria alternata) in complex biological matrices.
Control experiments with unlabeled PBA, heat-inactivated enzyme, and control strains showed no significant signal change, confirming assay specificity.

Conclusions:

5-FAM labeled polymers provide a sensitive and simple platform for high-throughput screening of polymer-degrading enzymes.
This method facilitates the discovery of novel microorganisms and enzymes capable of degrading synthetic polymers.
The fluorescent assay is adaptable for diverse environmental samples and enzyme sources, advancing bioremediation and recycling efforts.