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

Jie Zhang; Ruida Shan; Xia LI; Zhixiong Zeng; Dengyue Sun

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

Author: Jie ZHANG ¹ ; Ruida SHAN ¹ ; Xia LI ¹ ; Zhixiong ZENG ¹ ; Dengyue SUN ¹
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1. School of Biological Engineering, Qilu University of Technology (Shandong Academy of Sciences), Ji'nan 250000, Shandong, China.
Publication Type:Journal Article
Keywords: bis-(2-hydroxyethyl) terephthalate (BHET) degrading enzyme; enzymatic properties; isolation and purification; poly(ethylene terephthalate) (PET)
MeSH: Actinomycetales/genetics*; Hydrolases/metabolism*; Phthalic Acids/chemistry*; Polyethylene Terephthalates/metabolism*
From: Chinese Journal of Biotechnology 2023;39(5):2027-2039
CountryChina
Language:Chinese
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 ℃. 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 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.