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

Abstract

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-labeled 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 labeled 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 Pichia pastoris expressing the identical cutinase was cultivated in the presences of labeled 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 hydrolyzed labeled 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.