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.

作　　者：张洁[1] 单瑞达 李霞 曾志雄 孙登岳 ZHANG Jie;SHAN Ruida;LI Xia;ZENG Zhixiong;SUN Dengyue(School of Biological Engineering,Qilu University of Technology(Shandong Academy of Sciences),Ji’nan 250000,Shandong,China;State Key Laboratory of Bio-based Materials and Green Papermaking,Qilu University of Technology(Shandong Academy of Sciences),Ji’nan 250000,Shandong,China)

机构地区：[1]齐鲁工业大学(山东省科学院)生物工程学院,山东济南250000 [2]齐鲁工业大学(山东省科学院)生物基材料与绿色造纸国家重点实验室,山东济南250000

出　　处：《生物工程学报》2023年第5期2027-2039,共13页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(22207060);山东省自然科学基金(ZR2021QC193,ZR2021QC038)。

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℃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℃和8.0,在25–35℃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Co^(2+)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。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℃.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℃ and 8.0,and more than 80%of the enzyme activity could be maintained in the range of 25–35℃ and pH 7.0–9.0 and Co^(2+) 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.

关 键 词：聚对苯二甲酸乙二醇酯(PET) 双(2-羟乙基)对苯二甲酸酯(BHET)降解酶 分离纯化 酶学性质 

分 类 号：X705[环境科学与工程—环境工程]