一种新型聚酰胺水解酶的鉴定和性质分析

Identification and characterization of a novel polyamide hydrolase

摘要:

不可自然降解塑料的广泛应用对生态环境的危害日益严峻，塑料高聚物污染物已成为生态环境治理的焦点，其中利用酶等生物法对高聚物进行解聚具有一定优势，不仅反应条件温和，解聚产物还可以进行回收进入再循环。本研究使用4-硝基丙酰苯胺为模式底物，对实验室建立的塑料解聚酶库进行筛选，获得了一个来源于红色亚栖热菌(Meiothermus ruber)的能水解酰胺键的α/β水解酶MrABH，将其在大肠杆菌中进行表达，并利用亲和层析技术纯化获得了纯度较高的酶，对其催化性质、酶学性质以及催化聚酰胺的产物进行了研究，发现MrABH在pH 8.0–10.0都有良好的稳定性，其最适pH为9.0，最适温度为30 ℃；动力学分析表明其对酯键和酰胺键的催化效果相近，MrABH能解聚尼龙6 (polyamide 6, PA6)和尼龙66 (polyamide 66, PA66)生成单体和寡聚物，未来有望应用于聚酰胺的生物解聚和再循环利用。

关键词:聚酰胺;聚酰胺水解酶;表达纯化;酶学性质

Abstract:

The widespread use of non-naturally degradable plastics is causing increasingly serious harm to the environment. Reducing plastic pollutants has become the core of ecological and environment management. Biological methods such as enzymes demonstrate advantages in depolymerizing plastics with mild reaction conditions and recycling of depolymerization products. However, there are few reports on the biological depolymerization of polyamide plastics. In this study, by using 4-nitropropionanilide as the model substrate, we screened against our plastic depolymerase library and obtained a Meiothermus ruber-derived enzyme (MrABH) that can hydrolyze the polyamide bond. We expressed this enzyme in Escherichia coli and purified the protein by affinity chromatography. Furthermore, we investigated the catalytic properties, enzymatic properties, and catalytic products of this enzyme with polyamide as the substrate. MrABH had good stability at pH 8.0–10.0, with the optimal performance at pH 9.0 and 30 ℃. The catalytic performance of this enzyme for ester bonds and amide bonds was similar. MrABH can catalyze the depolymerization of PA6 and PA66 to produce monomers and oligomers, demonstrating the potential to be used in the depolymerization and recycling of polyamide.

Key words:polyamide;polyamide hydrolase;expression and purification;enzymatic properties