Bis(2‐hydroxyethyl) terephthalate (BHET) is an important compound produced from poly(ethylene terephthalate) (PET) cleavage. It was selected as the representative substance for the study of PET degradation. A bacterial strain HY1 that could degrade BHET was isolated and identified as Enterobacter sp. The optimal temperature and pH for BHET biodegradation were determined to be 30°C and 8.0, respectively. The half‐life of degradation was 70.20 h at an initial BHET concentration of 1,000 mg/L. The results of metabolites' analysis by liquid chromatograph–mass spectrometer revealed that BHET was first converted to mono‐(2‐hydroxyethyl) terephthalate (MHET) and then to terephthalic acid. Furthermore, an esterase‐encoding gene, estB, was cloned from strain HY1, and the expressed enzyme EstB was characterized. The esterase has a molecular mass of approximately 25.13 kDa, with an isoelectric point of 4.68. Its optimal pH and temperature were pH 8.0 and 40°C, respectively. The analysis of the enzymatic products showed that EstB could hydrolyze one ester bond of BHET to MHET. To the best of authors' knowledge, this is the first report on the biodegradation characteristics of BHET by a member of the Enterobacter genus.

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