Faculty of Applied Natural Sciences, TH Köln University of Applied Sciences - Leverkusen Campus, Leverkusen, Germany.
ICP, CSIC, Madrid, Spain.
Appl Microbiol Biotechnol. 2023 Apr;107(7-8):2209-2221. doi: 10.1007/s00253-023-12422-6. Epub 2023 Feb 21.
Biobased polymers derived from plant oils are sustainable alternatives to petro based polymers. In recent years, multienzyme cascades have been developed for the synthesis of biobased ω-aminocarboxylic acids, which serve as building blocks for polyamides. In this work, we have developed a novel enzyme cascade for the synthesis of 12-aminododeceneoic acid, a precursor for nylon-12, starting from linoleic acid. Seven bacterial ω-transaminases (ω-TAs) were cloned, expressed in Escherichia coli and successfully purified by affinity chromatography. Activity towards the oxylipin pathway intermediates hexanal and 12-oxododecenoic acid in their 9(Z) and 10(E) isoforms was demonstrated for all seven transaminases in a coupled photometric enzyme assay. The highest specific activities were obtained with ω-TA from Aquitalea denitrificans (TR), with 0.62 U mg for 12-oxo-9(Z)-dodecenoic acid, 0.52 U mg for 12-oxo-10(E)-dodecenoic acid and 1.17 U mg for hexanal. A one-pot enzyme cascade was established with TR and papaya hydroperoxide lyase (HPL), reaching conversions of 59% according to LC-ELSD quantification. Starting from linoleic acid, up to 12% conversion to 12-aminododecenoic acid was achieved with a 3-enzyme cascade comprising soybean lipoxygenase (LOX-1), HPL and TR. Higher product concentrations were achieved by the consecutive addition of enzymes compared to simultaneous addition at the beginning. KEY POINTS: • Seven ω-transaminases converted 12-oxododecenoic acid into its corresponding amine. • A three-enzyme cascade with lipoxygenase, hydroperoxide lyase, and ω-transaminase was established for the first time. • A one-pot transformation of linoleic acid to 12-aminododecenoic acid, a precursor of nylon-12 was achieved.
生物基聚合物由植物油衍生而来，是石油基聚合物的可持续替代品。近年来，已经开发出多酶级联反应来合成生物基ω-氨基羧酸，这些羧酸是聚酰胺的构建块。在这项工作中，我们从亚油酸出发，开发了一种用于合成尼龙-12 前体 12-氨基十二烯酸的新型酶级联反应。克隆了七种细菌ω-转氨酶（ω-TA），在大肠杆菌中表达并通过亲和层析成功纯化。在偶联的比色酶测定中，所有七种转氨酶都对其 9（Z）和 10（E）异构体的氧化脂肪族途径中间体己醛和 12-氧代十二烯酸表现出活性。ω-TA Aquitalea denitrificans（TR）的比活力最高，对于 12-氧代-9（Z）-十二烯酸为 0.62 U mg，对于 12-氧代-10（E）-十二烯酸为 0.52 U mg，对于己醛为 1.17 U mg。用 TR 和木瓜过氧化物酶（HPL）建立了一锅酶级联反应，根据 LC-ELSD 定量达到 59%的转化率。从亚油酸开始，通过包含大豆脂氧合酶（LOX-1）、HPL 和 TR 的三酶级联反应，可实现 12-氨基十二烯酸 12%的转化率。与同时添加相比，连续添加酶可获得更高的产物浓度。关键点：• 七种 ω-转氨酶将 12-氧代十二烯酸转化为其相应的胺。• 首次建立了包含脂氧合酶、过氧化物酶和 ω-转氨酶的三酶级联反应。• 实现了从亚油酸到尼龙-12 前体 12-氨基十二烯酸的一锅转化。
