工程菌过氧化物酶降解热固性复合环氧树脂的比色高通量筛选

M. Dolz, Ivan Mateljak, D. Méndez-Sánchez, Israel Sánchez-Moreno, Patricia Gomez de Santos, Javier Viña‐Gonzalez, M. Alcalde
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引用次数: 2

摘要

目前,热固性复合环氧树脂的报废管理仅限于焚烧和垃圾填埋储存,这突出了对制定更可持续措施的需求。真菌非特异性过氧酶(UPOs)由于其广泛的C-H氧官能化反应,正成为有机合成中重要的生物技术工具,而其在生物降解过程中的潜在用途不容低估。在这里,我们提出了一种比色筛选方法,旨在工程UPOs降解环氧树脂。我们的研究基于Hexflow®RTM-6,这是一种在航空领域广泛使用的商业环氧树脂。来自短家族和长家族的UPO突变体最初通过GC/MS进行基准测试,以确定其对Hexflow®RTM-6的主要结构支架N,N-双(2-羟丙基)-对甲苯苯胺(NNBT)的潜在N-脱烷基活性。建立了一种可靠的高通量比色筛选方法来定量UPO攻击NNBT叔胺释放的乳醛。基于酵母表达的阿伯达Psathyrella aberdarensis进化的UPO,构建了一小部分具有不同突变负载量的突变文库,并对其进行了NNBT N-脱烷基筛选,从而建立了一个定向进化平台,作为设计UPO复合降解变体的载体。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Colorimetric High-Throughput Screening Assay to Engineer Fungal Peroxygenases for the Degradation of Thermoset Composite Epoxy Resins
At present, the end-of-life management of thermoset composite epoxy resins is limited to incineration and landfill storage, highlighting the demand for the development of more sustainable measures. Due to their broad spectrum of C-H oxyfunctionalization reactions, fungal unspecific peroxygenases (UPOs) are becoming important biotechnological tools in organic synthesis while their potential use in biodegradation processes should not be underestimated. Here, we present a colorimetric screening assay aimed at engineering UPOs for the degradation of epoxy resins. We based our study on Hexflow® RTM-6, a commercial epoxy resin used extensively in the aeronautics sector. UPO mutants from the short and long families were initially benchmarked by GC/MS to determine their potential N-dealkylation activity on N,N-bis(2-hydroxypropyl)-p-toluidine (NNBT), the main structural scaffold of Hexflow® RTM-6. A reliable high-throughput colorimetric screening method was developed to quantify the lactaldehyde released by UPO attack on the tertiary amine of NNBT. Based on an evolved UPO from Psathyrella aberdarensis that was expressed by yeast, a small subset of mutant libraries with different mutational loadings was constructed and screened for NNBT N-dealkylation, thereby establishing a directed evolution platform as a vehicle to engineer UPO composite degrading variants.
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