Agar plate-based screening methods for the identification of polyester hydrolysis by Pseudomonas species

Abstract

Hydrolases acting on polyesters like cutin, polycaprolactone or polyethylene terephthalate (PET) are of interest for several biotechnological applications like waste treatment, biocatalysis and sustainable polymer modifications. Recent studies suggest that a large variety of such enzymes are still to be identified and explored in a variety of microorganisms, including bacteria of the genus Pseudomonas. For activity-based screening, methods have been established using agar plates which contain nanoparticles of polycaprolactone or PET prepared by solvent precipitation and evaporation. In this protocol article, we describe a straightforward agar plate-based method using emulsifiable artificial polyesters as substrates, namely Impranil^® DLN and liquid polycaprolactone diol (PLD). Thereby, the currently quite narrow set of screening substrates is expanded. We also suggest optional pre-screening with short-chain and middle-chain-length triglycerides as substrates to identify enzymes with lipolytic activity to be further tested for polyesterase activity. We applied these assays to experimentally demonstrate polyesterase activity in bacteria from the P. pertucinogena lineage originating from contaminated soils and diverse marine habitats.

Figure 1

Workflow for agar plate‐based screening for polyesterase active clones. A. Steps of plate preparation and screening: 1. Prepare an emulsion/suspension with the respective substrate (if necessary). 2. Combine substrate emulsion/suspension and molten agar‐containing nutrient medium. 3. Pour the warm medium into suitable Petri dishes and let the agar solidify. Suitable supplements for induction of gene expression or selection may be included as well. 4. Plate bacteria either by transfer of single colonies using autoclaved toothpicks, 96 pin replicators or a robotic colony picker, or spread appropriate cell suspensions with glass beads or a Drigalski spatula. Incubate for at least 16 h at a temperature optimal for the applied organism. 5. Document the appearance of halos and/or fluorescence if applicable. B. Overview on the described substrates (including the chain lengths of the dominant fatty acid for the triacylglycerides) and the enzymatic activities that can be identified with the respective screening plates.

Figure 2

Polyesterase activities exhibited by Pseudomonas species. The colonies were grown for 24 h at 30°C on LB agar plates supplemented with different substrates: Tributyrin (esterase activity); coconut oil + rhodamine B (mid‐chain‐length hydrolyzing esterase); Impranil^® DLN (synthetic polyester polyurethane, polyesterase activity); PCD_{M n530}, polycaprolactone diol (synthetic polyester, polyesterase activity); and polycaprolactone nanoparticles (current standard for polyesterase screens, polyesterase activity). P. putida as an example for a fluorescent Pseudomonad and E. coli as a negative control are indicated by grey letters. The white halo around P. putida relies on the fluorescence of the siderophore pyoverdine and does not indicate clearance of the substrate. All plates were photodocumented under white light, except coconut oil + rhodamine B‐supplemented plates which were exposed to UV light (λ = 254 nm). Shown are exemplary colonies of a set of at least three colonies for each combination on independent plates. Halo formation of the depicted colony is representative for all replicates.