Chen_2023_Int.J.Mol.Sci_24_2780

Poly(ethylene terephthalate) (PET) is a manufactured plastic broadly available, whereas improper disposal of PET waste has become a serious burden on the environment. Leaf-branch compost cutinase (LCC) is one of the most powerful and promising PET hydrolases, and its mutant LCC(ICCG) shows high catalytic activity and excellent thermal stability. However, low binding affinity with PET has been found to dramatically limit its further industrial application. Herein, TrCBM and CfCBM were rationally selected from the CAZy database to construct fusion proteins with LCC(ICCG), and mechanistic studies revealed that these two domains could bind with PET favorably via polar amino acids. The optimal temperatures of LCC(ICCG)-TrCBM and CfCBM-LCC(ICCG) were measured to be 70 and 80 degreesC, respectively. Moreover, these two fusion proteins exhibited favorable thermal stability, maintaining 53.1% and 48.8% of initial activity after the incubation at 90 degreesC for 300 min. Compared with LCC(ICCG), the binding affinity of LCC(ICCG)-TrCBM and CfCBM-LCC(ICCG) for PET has been improved by 1.4- and 1.3-fold, respectively, and meanwhile their degradation efficiency on PET films was enhanced by 3.7% and 24.2%. Overall, this study demonstrated that the strategy of constructing fusion proteins is practical and prospective to facilitate the enzymatic PET degradation ability.