来源于糖丝菌双(2-羟乙基)对苯二甲酸酯水解酶的酶学性质表征及降解特性分析

Enzymatic properties and degradation characterization of a bis(2-hydroxyethyl) terephthalate hydrolase from Saccharothrix sp.

摘要:

聚对苯二甲酸乙二醇酯[poly(ethylene terephthalate), PET]降解酶的发掘是国内外研究的热点。双(2-羟乙基)对苯二甲酸酯[bis-(2-hydroxyethyl) terephthalic acid, BHET]是PET降解过程的一种中间化合物，会与PET竞争酶的底物结合位点，从而抑制PET进一步降解。因此，探寻新型BHET降解酶，对进一步提高PET的降解效率具有促进作用。本研究通过基因挖掘发现了一种来源于浅黄糖丝菌(Saccharothrix luteola)参与PET降解过程的水解酶基因sle (ID: CP064192.1, 5085270-5086049)，其编码的蛋白质可以将BHET水解为单(2-羟乙基)对苯二甲酸酯[mono-(2-hydroxyethyl) terephthalate, MHET]和对苯二甲酸(terephthalic acid, TPA)。将BHET水解酶(Sle)通过重组质粒在大肠杆菌(Escherichia coli)中异源表达，结果表明，在异丙基-β-d-硫代半乳糖苷(isopropyl-β-d-thiogalactoside, IPTG)诱导终浓度为0.4 mmol/L，诱导时长为12 h，诱导温度为20 °C时蛋白的表达量最高。通过镍亲和层析、阴离子交换层析和凝胶过滤层析3步分离纯化，获得了高纯度的Sle重组蛋白；同时对其酶学性质进行了表征，Sle最适温度和pH分别为35 °C和8.0，在25-35 °C和pH 7.0-9.0区间内能保持80%以上的残余酶活，且金属离子Co²⁺能提高酶活力；进一步通过同源序列及Sle复合物结构分析得知，该酶属于二烯酸内酯水解酶(dienelactone hydrolase, DLH)家族，具备该家族典型的催化三联体，预测其催化位点分别为S129、D175和H207，并初步分析了其催化机理。最后，利用高效液相色谱法(high performance liquid chromatography, HPLC)鉴定了该酶能够特异性降解BHET生成MHET和TPA，属于BHET降解酶。本研究为生物酶法高效降解PET塑料提供了新的酶资源。

Abstract:

The discovery of new enzymes for poly(ethylene terephthalate) (PET) degradation has been a hot topic of research globally. Bis-(2-hydroxyethyl) terephthalate (BHET) is an intermediate compound in the degradation of PET and competes with PET for the substrate binding site of the PET-degrading enzyme, thereby inhibiting further degradation of PET. Discovery of new BHET degradation enzymes may contribute to improving the degradation efficiency of PET. In this paper, we discovered a hydrolase gene sle (ID: CP064192.1, 5085270-5086049) from Saccharothrix luteola, which can hydrolyze BHET into mono-(2-hydroxyethyl) terephthalate (MHET) and terephthalic acid (TPA). BHET hydrolase (Sle) was heterologously expressed in Escherichia coli using a recombinant plasmid, and the highest protein expression was achieved at a final concentration of 0.4 mmol/L of isopropyl-β-d-thiogalactoside (IPTG), an induction duration of 12 h and an induction temperature of 20 °C. The recombinant Sle was purified by nickel affinity chromatography, anion exchange chromatography, and gel filtration chromatography, and its enzymatic properties were also characterized. The optimum temperature and pH of Sle were 35 °C and 8.0, and more than 80% of the enzyme activity could be maintained in the range of 25-35 °C and pH 7.0-9.0 and Co²⁺ could improve the enzyme activity. Sle belongs to the dienelactone hydrolase (DLH) superfamily and possesses the typical catalytic triad of the family, and the predicted catalytic sites are S129, D175, and H207. Finally, the enzyme was identified as a BHET degrading enzyme by high performance liquid chromatography (HPLC). This study provides a new enzyme resource for the efficient enzymatic degradation of PET plastics.