Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, Forschungszentrum Jülich, D-52425, Jülich, Germany.
Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, D-52425, Jülich, Germany.
Microb Biotechnol. 2020 Jan;13(1):274-284. doi: 10.1111/1751-7915.13418. Epub 2019 Apr 23.
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.
水解酶作用于聚酯，如角质、聚己内酯或聚对苯二甲酸乙二醇酯（PET），在废物处理、生物催化和可持续聚合物改性等多个生物技术应用中具有重要意义。最近的研究表明，在包括假单胞菌属细菌在内的各种微生物中，仍有大量此类酶有待发现和探索。为了进行基于活性的筛选，已经建立了使用琼脂平板的方法，其中包含通过溶剂沉淀和蒸发制备的聚己内酯或 PET 的纳米颗粒。在本方案文章中，我们描述了一种使用可乳化人工聚酯作为底物的简单琼脂平板方法，即 Impranil DLN 和液体聚己内酯二醇（PLD）。由此，扩展了目前相当狭窄的筛选底物集。我们还建议使用短链和中链长甘油三酯作为底物进行可选的预筛选，以鉴定具有脂肪酶活性的酶，然后进一步测试其聚酯酶活性。我们应用这些测定法从源自污染土壤和各种海洋生境的 pertucinogena 谱系假单胞菌属细菌中实验性地证明了聚酯酶活性。
