Available online 5 April 2025

A novel method for detecting polymer hydrolysis in complex matrices was developed by covalently coupling 5-carboxy-fluorescein (5-FAM) to poly(butylene adipate) (PBA) and blending it at varying concentrations.

Thc_Cut1 cutinase from Thermobifida cellulosilytica demonstrated efficient hydrolysis of 5-FAM-labelled PBA blends, resulting in a fluorescence increase, confirming its enzymatic activity.

The method enabled fluorescence-based detection of polymer hydrolysis by Pichia pastoris expressing Thc_Cut1 in a complex matrix, showcasing potential for high-throughput screening of polymer-decomposing enzymes and microbes.

Despite recent advances, there is still a demand for more efficient enzymes hydrolyzing synthetic polymers. Automated high throughput screening strategies of microorganisms from different environments could yield novel enzymes but require specific methods for detection of polymer hydrolysis in complex matrices. Here, 5-carboxy-fluorescein (5-FAM) was covalently coupled to poly(butylene adipate) (PBA) and blended at 1%, 5% and 10% w/w concentrations with non-labelled PBA. Hydrolysis of PBA by the Thc_Cut1 cutinase from Thermobifida cellulosilytica was confirmed via quantification of the released monomers 1,4-butanediol and adipic acid, weight loss and FTIR analysis. Upon incubation with Thc_Cut1, hydrolysis of all three fluorescent labelled PBA blends lead to a clear fluorescence increase of up to 4000 RFU while no signal change was detected for the blank and for heat-inactivated enzyme (signal below 500 RFU). In a next step, as a model organism Picha pastoris expressing the identical cutinase was cultivated in the presences of labelled PBA. Despite the complex matrix, a fluorescence increase of up to 500 RFU was observed for P. pastoris expressing the enzyme while no significant signal change was seen for the control strain (lacking Thc_Cut1 expression). Likewise, extracellular enzymes from the fungi Fusarium solani and Alternaria alternata hydrolysed labelled PBA leading to fluorescence increases of 1328 and 1187 RFU. This indicates that 5-FAM covalently coupled to polymers could be used for development of simple and high throughput screening platforms to identify polymer decomposing microorganisms and enzymes.

High-throughput screening

5-carboxy-fluorescein (5-FAM)

Poly(butylene adipate) (PBA)

polymer-degrading enzymes

Pichia Pastoris

© 2025 The Author(s). Published by Elsevier B.V.