Preparation and hydrolytic activity analysis of dual-catalytic-triad PETase
- VernacularTitle:双催化三联体PET水解酶的制备及其酶活性分析
- Author:
Qiudong SU
1
;
Xining YAO
2
;
Feng QIU
1
;
Feng WANG
1
;
Shuang ZHANG
1
;
Ke XU
1
;
Shengli BI
1
;
Yanhai WANG
1
Author Information
- Publication Type:Journal Article
- Keywords: polyethylene terephthalate; hydrolase; dual catalytic triad; site-directed mutagenesis; recombinant protein; biodegradation
- From: Acta Universitatis Medicinalis Anhui 2026;61(3):546-551
- CountryChina
- Language:Chinese
- Abstract: ObjectiveTo prepare a recombinant PETase with a dual-catalytic-triad and to evaluate its efficiency in the biodegradation of polyethylene terephthalate （PET）. MethodsBased on the crystal structure of wild-type PETase， point mutations （T88H/L117D） were introduced via site-directed mutagenesis. The recombinant protein was prepared using prokaryotic expression and chromatography purification techniques. The enzymatic hydrolysis of the mutant PETase was assessed by relatively quantifying the products mono （2-hydroxyethyl） terephthalate （MHET） and terephthalic acid （TPA）. ResultsBoth wild-type and mutant PETases accumulated as inclusion bodies， accounting for approximately 20% of the total bacterial protein. After solubilization in urea， the proteins were eluted at 300 mmol/L imidazole during affinity chromatography purification， with concentrations of 1.824 and 1.833 mg/mL and purities of 83.11% and 84.32%， respectively. Subsequent anion-exchange chromatography yielded highly pure enzymes in the 200 mmol/L NaCl fraction： 2.776 mg/mL （96.86% purity） for the wild type and 1.967 mg/mL （95.13% purity） for the mutant. Following refolding， the final concentrations were 0.484 mg/mL for the wild type and 0.991 mg/mL for the mutant. Hydrolysis assays revealed that the mutant released MHET and TPA at （237.67±17.00）% and （197.33±12.01）% of the wild-type levels， respectively. ConclusionThe T88H/L117D dual-catalytic-triad PETase is successfully prepared and it significantly enhanced PET-degrading activity， thus， it′s a promising biocatalyst for PET bioremediation.
