Study On The Natural Evolutionary Protein Engineering Of Stable Ispetase For PET Plastic Degradation

Poly(ethylene terephthalate)(PET)is used widely by human beings,but is very difficult to degrade,resulting in widespread distribution in the biosphere,and causing a series of ecological and social problems.Up to now,the PET degradation effect of PETase from Ideonella sakaiensis 201-F6(IsPETase)variants with low stability and activity was not ideal,especially for highly-crystalline PET.In this study,a mutation design tool,Premuse,was developed to integrate the sequence alignment and quantitative selection of the preferred mutations based on natural sequence evolution from a large number of homologs.Ten single point mutants were selected from 1,486 homologous sequences using Premuse,and then two mutations(W159H and F229Y)with improved stability were screened from them.The derived double point mutant,W159H/F229Y,exhibited a strikingly enhanced enzymatic performance.Its Tm and catalytic efficiency values(kcat/Km)respectively increased by 10.4? and 1.5-fold using p-NPP as the substrate compared with wild type.The degradation activity for amorphous PET was increased by almost 40-fold in comparison with wild type at 40? in 24h.Additionally,the result of PET degradation showed that the PET bottle waste was obviously biodegraded and reduced by W159H/F229Y in 24h.This study allowed us to more efficiently and exquisitely design the mutation based on natural evolution,and provides a tool for achieving biodegradation of PET pollution under mild natural environments.