Attack Site Density of a Highly-efficient PET Hydrolases

Abstract

Introduction: Poly (ethylene terephthalate) (PET) is one of the most abundant polyester materials used in daily life and it is also one of the main culprits of environmental pollution. ICCG (F243I/D238C/S283C/Y127G) is an enzyme that performs four modifications on the leaf branch compost keratase (LCC). It shows excellent performance in the hydrolysis of PET and has a great potential in further applications. Methods: Here, we used ICCG to degrade PET particles of various sizes and use the density of attack sites (Γattack) and kinetic parameters to evaluate the effect of particle size on enzyme degradation efficiency. We are surprised to observe that there is a certain relationship between Km and Γattack. In order to further confirm the relationship, we obtained three different enzymes (Y95K, M166S and H218S) by site-directed mutagenesis on the basis of ICCG. Result: The results confirmed that there was a negative correlation between Km and Γattack. In addition, we also found that increasing the affinity between the enzyme and the substrate does not necessarily lead to the increase of degradation rate. Conclusion: These findings show that the granulation of PET and the selection of appropriate particle size are helpful to improve its industrial application value. At the same time, additional protein engineering to increase ICCG performance is realistic, but it can’t be limited to enhance the affinity between enzyme and substrate.