A Coupled Ketoreductase‐Diaphorase Assay for the Detection of Polyethylene Terephthalate‐Hydrolyzing Activity

Abstract

In the last two decades, several PET‐degrading enzymes from already known microorganisms or metagenomic sources have been discovered to face the growing environmental concern of polyethylene terephthalate (PET) accumulation. However, there is a limited number of high‐throughput screening protocols for PET‐hydrolyzing activity that avoid the use of surrogate substrates. Herein, a microplate fluorescence screening assay was described. It was based on the coupled activity of ketoreductases (KREDs) and diaphorase to release resorufin in the presence of the products of PET degradation. Six KREDs were identified in a commercial panel that were able to use the PET building block, ethylene glycol, as substrate. The most efficient KRED, KRED61, was combined with the diaphorase from Clostridium kluyveri to monitor the PET degradation reaction catalyzed by the thermostable variant of the cutinase‐type polyesterase from Saccharomonospora viridis AHK190. The PET degradation products were measured both fluorimetrically and by HPLC, with excellent correlation between both methods.

The Impact of Metagenomics on Biocatalysis

“…[58] Consequently, many companies have invested in commercial panels of KREDs from known or metagenomic sources along with assays to screen large libraries at once. [59] Newgas et al isolated a panel of KREDs from an in silico oral microbiome metagenomic library, which demonstrated excellent activity on a number of sterically hindered ketones. [60] It has also been shown that metagenomic KREDs can be used to make synthetically challenging drug fragments.…”

“…Ketoreductases (KREDs) or alcohol dehydrogenases (ADHs) are able to the reduction of ketones in a wide range of different molecules [58] . Consequently, many companies have invested in commercial panels of KREDs from known or metagenomic sources along with assays to screen large libraries at once [59] . Newgas et al.…”

Sub-single-turnover quantification of enzyme catalysis at ultrahigh throughput via a versatile NAD(P)H coupled assay in microdroplets

“…Leakage is a well-documented challenge in microfluidic assay development, preventing the use of the commonly large and aromatic probes for all but the fastest reactions [48][49][50][51][52] . This poses a hitherto unsolved challenge for fluorogenic NAD(P)H quantification as resorufin, the sole published fluorogenic probe for NAD(P)H detection [49,53] , is hydrophobic and consequently only compatible with very short reaction times in microfluidic droplets [54] . Previous work has shown that adding charged groups to fluorophores [52] and specifically coumarins [12,55] mitigates droplet leakage and improves aqueous solubility.…”

New Labeled PET Analogues Enable the Functional Screening and Characterization of PET-Degrading Enzymes

“…For instance, bulk UV absorbance measurement of the reaction supernatant has been proposed as a simpler method, but it comes with some disadvantages: depending on the mode of action of the enzyme (if it produces monomers or oligomers), the measurements can be inaccurate and difficult to compare . Alternatively, the utilization of ketoreductases (KREDs) for the detection of released ethylene glycol (EG) showed a good correlation to the HPLC results . Another approach for quantifying PET degradation products, initially introduced by Ebersbach et al, converts products into fluorescent compounds through a mechanism driven by the iron autoxidation-mediated generation of free hydroxyl radicals .…”

“… 14 Alternatively, the utilization of ketoreductases (KREDs) for the detection of released ethylene glycol (EG) showed a good correlation to the HPLC results. 15 Another approach for quantifying PET degradation products, initially introduced by Ebersbach et al, converts products into fluorescent compounds through a mechanism driven by the iron autoxidation-mediated generation of free hydroxyl radicals. 16 However, Zurier and Goddard highlighted that this particular protocol encounters limitations that arise from the fact that the mono-(2-hydroxyethyl) terephthalate (MHET) exhibits a lower fluorescent extinction coefficient compared to that of terephthalic acid (TPA), 13 so in a hydrolysis mixture where both products are present, TPA will contribute mostly to fluorescence.…”