Agar plate-based screening methods for the identification of polyester hydrolysis by Pseudomonas species.,Microbial Biotechnology

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Agar plate-based screening methods for the identification of polyester hydrolysis by Pseudomonas species.
Microbial Biotechnology ( IF 4.8 ) Pub Date : 2019-04-23 , DOI: 10.1111/1751-7915.13418
Rebecka Molitor ₁ , Alexander Bollinger ₁ , Sonja Kubicki ₁ , Anita Loeschcke ₁ , Karl-Erich Jaeger _{1,

2} , Stephan Thies ₁

Affiliation

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.

中文翻译：

用于鉴定假单胞菌属聚酯水解的琼脂平板筛选方法。

作用于角质、聚己内酯或聚对苯二甲酸乙二醇酯 (PET) 等聚酯的水解酶引起了废物处理、生物催化和可持续聚合物改性等多种生物技术应用的兴趣。最近的研究表明，在包括假单胞菌属细菌在内的多种微生物中，仍有大量此类酶有待鉴定和探索。对于基于活性的筛选，已经建立了使用琼脂板的方法，该琼脂板含有通过溶剂沉淀和蒸发制备的聚己内酯或 PET 纳米颗粒。在这篇协议文章中，我们描述了一种基于琼脂平板的简单方法，使用可乳化的人造聚酯作为底物，即 Impranil ^® DLN 和液体聚己内酯二醇 (PLD)。从而，扩大了目前相当狭窄的筛选基底组。我们还建议可选地以短链和中链甘油三酯作为底物进行预筛选，以鉴定具有脂肪分解活性的酶，以进一步测试聚酯酶活性。我们应用这些测定法通过实验证明来自受污染土壤和不同海洋栖息地的P. pertucinogena谱系的细菌中的聚酯酶活性。

更新日期：2019-04-23

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