Rational Modification Of Leaf-branch Composite Cutinase For PET Plastic Degradation

Plastic is a synthetic polymer compound,mainly made from petrochemical raw materials,with high molecular weight and plasticity.Poly（ethylene terephthalate）（PET）is one of the representative plastics used in our daily life.PET is a synthetic polyester that is linked by terephthalic acid（TPA）and ethylene glycol（EG）through ester bonds.Because of its strong plasticity and durability,it is widely used in all aspects of life.However,PET waste plastics produced after use and accumulated in large quantities in the environment have posed a serious threat to the ecological environment.PET waste has many treatment methods,but it is easy to cause more waste of resources and secondary pollution.With the development of biotechnology,the biodegradation of plastic waste has a good application prospect in the treatment of PET waste.In this study,ICCG（a quadruple mutant of the metagenomic derived enzyme LCC,F243I/D238C/S283C/N246G）with high PET hydrolysis activity was used as the research object.Mono（2-hydroxyethyl）terephthalic acid（MHET）was used as the substrate to analyze the basic physical and chemical properties of ICCG.The optimum p H of ICCG was 5.0,the optimum temperature was 70°C,and the stability was good.Considering that the hydrolysis process of PET is a heterogeneous catalytic process,the enzymatic kinetic properties of ICCG on intermediate products MHET and BHET were analyzed in order to understand the competitive inhibition of intermediate products on ICCG.Further hydrolysis of MHET was determined as the main rate-limiting step.Compared with the complete PET material,PET particles with larger specific surface area are more suitable for biocatalytic degradation.Using ICCG to degrade PET plastic particles,the optimal PET particle size in the reaction process was determined to be 300μm in combination with the Mi equation model and the actual industrial production..In this process,the substrate inhibition phenomenon was found.In order to better screen the particle size,the substrate inhibition of PET particles on ICCG was determined under various particle size conditions,and the optimal reaction particle size of PET was determined to be 300μm.In order to further improve the catalytic activity of ICCG on PET,the basic sequence alignment and structural analysis of ICCG were carried out by bioinformatics methods.The mutants of ICCG Y95K,M166S,H218S,Y95K-M166S and H218S-F222I were obtained by site-directed mutagenesis.Attack site density(Γ_attack)was introduced to evaluate multiple mutants by measuring the optimal temperature,combining with the Michalis equation and substrate inhibition constant.The affinity of the mutant Y95K to PET plastic particles has been greatly improved,and the mutant M166S to Vmax has increased.Most notably,the optimum temperature of the double mutant H218S-F222I is 50°C,and the catalytic rate of PET at the optimum temperature is 4 times that of ICCG at the same temperature,although the stability was reduced at higher temperatures.The results of this paper provide a basis for the transformation and production of PET hydrolase ICCG,which provides a basis for the largescale application of new PET hydrolase in industry.