来源于嗜热氢化杆菌的新型对苯二甲酸双(羟乙)酯水解酶的表达纯化与酶学性质

Expression, purification and characterization of a novel bis (hydroxyethyl) terephthalate hydrolase from Hydrogenobacter thermophilus

摘要:

石化来源的聚对苯二甲酸乙二酯(polyethylene terephthalate, PET)被广泛用于矿泉水瓶、食品包装和纺织品等领域，因其在自然界中不易分解，大量使用后的PET废弃物造成了严重的环境污染与资源浪费。使用生物酶法对PET废弃物进行解聚，并对解聚产物进行升级循环利用是进行塑料污染治理的重要方向之一，其中关键的是PET水解酶的解聚效率。对苯二甲酸双(羟乙基)酯(bis (hydroxyethyl) terephthalate, BHET)是PET生物酶解的中间产物，其累积是限制PET水解酶催化效率的一个重要因素，BHET水解酶和PET水解酶的联用能提升PET的整体水解效率。来源于嗜热氢化杆菌(Hydrogenobacter thermophilus)的双烯内酯酶(HtBHETase)对BHET有显著水解效果，将该酶在大肠杆菌(Escherichia coli)中进行重组表达并纯化后，对其酶学性质进行了研究。结果显示，HtBHETase对短碳链的酯类如对硝基苯酚乙酸酯催化活性较高，HtBHETase以BHET为底物时的最适反应pH值和最适反应温度分别为5.0和55 °C；该酶有较好的热稳定性，经80 °C的条件处理1 h仍能保持80%以上活性，显示出了良好的热稳定性，HtBHETase有在PET塑料生物解聚中使用的潜力，本研究为推动生物酶法降解PET提供了新的参考。

关键词:对苯二甲酸双(羟乙基)酯;水解酶;表达纯化;酶学性质

Abstract:

PET (polyethylene terephthalate) is one of the most important petrochemicals that is widely used in mineral water bottles, food and beverage packaging and textile industry. Because of its stability under environmental conditions, the massive amount of PET wastes caused serious environmental pollution. The use of enzymes to depolymerize PET wastes and upcycling is one of the important directions for plastics pollution control, among which the key is the depolymerization efficiency of PET by PET hydrolase. BHET (bis(hydroxyethyl) terephthalate) is the main intermediate of PET hydrolysis, its accumulation can hinder the degradation efficiency of PET hydrolase significantly, and the synergistic use of PET hydrolase and BHET hydrolase can improve the PET hydrolysis efficiency. In this study, a dienolactone hydrolase from Hydrogenobacter thermophilus which can degrade BHET (HtBHETase) was identified. After heterologous expression in Escherichia coli and purification, the enzymatic properties of HtBHETase were studied. HtBHETase shows higher catalytic activity towards esters with short carbon chains such as p-nitrophenol acetate. The optimal pH and temperature of the reaction with BHET were 5.0 and 55 °C, respectively. HtBHETase exhibited excellent thermostability, and retained over 80% residual activity after treatment at 80 °C for 1 hour. These results indicate that HtBHETase has potential in biological PET depolymerization, which may facilitate the enzymatic degradation of PET.

Key words:bis(hydroxyethyl) terephthalate (BHET);hydrolase;expression and purification;enzymatic properties