一种聚对苯二甲酸乙二醇酯(PET)水解新酶的挖掘与酶学性质分析

Discovery and characterization of a new enzyme for hydrolyzing polyethylene terephthalate (PET)

摘要:

目的 聚对苯二甲酸乙二醇酯(polyethylene terephthalate, PET)的生物酶法解聚是通过PET水解酶催化PET水解，得到终产物对苯二甲酸和乙二醇。通过基因挖掘获得新的PET水解酶，可为废弃PET的生物酶法解聚提供可用于分子改造的蛋白。方法 通过基因挖掘的方法发现来源于Alkalilimnicola ehrlichii的未知功能蛋白(命名为AePETase)具有水解无定形PET膜的活性，并对AePETase进行异源表达、纯化和酶学性质研究。结果 确定了AePETase的T_m值为(53.0±0.7) ℃，最适反应温度为45 ℃，最适缓冲液体系为Na₂HPO₄-NaH₂PO₄ (100 mmol/L, pH 8.0)，较合适的酶浓度为1.0 μmol/L以内。在45 ℃条件下，无定形PET膜的水解产物量为已报道的IsPETase的13.4倍(72 h)。结论 AePETase对PET具有较好的水解活性，是一种具有较大潜力的PET水解新酶。

Abstract:

Objective The enzymatic depolymerization of polyethylene terephthalate (PET) involves the hydrolysis of PET catalyzed by PET hydrolases, leading to the production of terephthalic acid and ethylene glycol. In this study, we identified a new PET hydrolase through gene mining, with the goal of providing enzymes for efficient depolymerization of waste PET.Methods A previously uncharacterized functional enzyme, named as AePETase, was identified through gene mining. This enzyme demonstrated the ability to hydrolyze amorphous PET films. Subsequently, heterologous expression, purification, and enzymatic characterization were performed for AePETase.Results AePETase showed a T_m value of (53.0±0.7) ℃, and an optimal reaction temperature of 45 ℃. The optimal buffer was Na₂HPO₄-NaH₂PO₄ (100 mmol/L, pH 8.0), and the optimal enzyme concentration was 1 μmol/L. After 72 h, the hydrolysis of amorphous PET films by AePETase resulted in 13.4-fold more products than that by the reported IsPETase.Conclusion AePETase demonstrates significant hydrolytic activity on PET and represents a promising new enzyme for hydrolyzing PET.