Accelerating The Degradation Of PET Plastic By Ultrasound Assisting And Molecular Modification Of Cutinase LCC

Polyethylene terephthalate(PET),a typical linear thermoplastic resin material,is widely used in manufacture of fiber,film and beverage bottle.Although PET brings many conveniences to human life,the white pollution has occurred and seriously threatened the global environment because of its difficult degradation property.Recently,researchers obtained a thermostable cutinase(LCC)by molecular modification which can degrade PET rapidly.In this study,the LCC was recombined and expressed in E.coli BL21(DE3).The enzymatic properties of LCC were studied,and physical(ultrasonic)and biological(fusion binding module)methods were used to promote the degradation of PET by recombinant cutinase LCC.The main research results are listed as follows:Recombinant E.coli BL21(DE3)-p ET-26b(+)-LCC was successfully constructed and used for overexpression of cutinase LCC.For protein expression,the recombinant expression strains were cultured at 37 °C in Luria-Bertani(LB)medium containing 50 ?g/m L kanamycin to an OD600 of 0.6-0.8,and then induced(16 °C,20 h)with 0.2 m M IPTG.Subsequently,the purified LCC was obtained by Ni-affinity chromatography,and the activity of the purified LCC was about 320 U/mg.The optimum temperature of recombinant LCC was 65 ? and the optimum p H was 8.0.The effect of ultrasonic power and time on the degradation of PET by recombinant LCC was studied.The optimal ultrasonic condition was 25 W ultrasonic power for 5 hours,and the degradation efficiency was 1.21 times higher than that of the non ultrasonic after 24 hours of degradation.The secondary structure of recombinant LCC was analyzed by circular dichroism and Fourier transform infrared spectroscopy.It was found that ultrasound made the structure of recombinant cutinase LCC more disordered,and the properties of the recombinant LCC changed accordingly.The SEM result showed that ultrasound promoted the degradation of PET by LCC.The expression strains E.coli BL21(DE3)-p ET-26b(+)-LCC-CBM and E.coli BL21(DE3)-p ET-26b(+)-LCC-PBM were successfully obtained.The effects of fusion binding module CBM and PBM on the degradation of PET by recombinant cutinase were studied.At24 hours,the degradation efficiency of LCC-PBM and LCC-CBM on PET was 2.1-fold and1.6-fold of that recombinant LCC,respectively.The SEM result showed that the fusion binding module promoted the degradation of PET by recombinant LCC.