Study On Whole-cell Catalytic Hydrolysis Of PET Plastics By E.Coli Based On Cell Surface Display Technology

Polyethylene terephthalate(PET)is the world’s most abundant polyester plastic with excellent properties and is widely used in many fields such as food packaging,chemicals,daily necessities,and mechanical and electronic applications.However,the stable and difficult to hydrolyze properties of PET also cause a huge burden on the environment,and its continuous accumulation in nature is causing global environmental problems and threatening human survival.At present,the main treatment methods of PET are still physical and chemical methods,but it has been criticized for the problem of secondary environmental pollution and high energy consumption.Enzymatic hydrolysis of waste PET,a green and environmentally friendly treatment method,has attracted more and more attention.However,the current biological enzymes that can be used for hydrolysis of PET have problems such as low activity,poor heat resistance,and unclear catalytic mechanism,which limit their application in industry.Therefore,it is of great significance to develop more excellent PET hydrolases and the engineering application of PET hydrolases.In this paper,three anchored proteins were linked to four PET hydrolases by cell surface display technology,and their hydrolysis efficiency of PET plastics was compared to optimize the best surface display recombinant protein system pGSA-FAST-PETase.On this basis,the enzymatic properties and hydrolysates of pGSA-FAST-PETase were further studied.However,the hydrolysate of FAST-PETase to PET plastics is mainly mono-(2-hydroxyethyl)terephthalic acid(MHET),in order to further open up the hydrolysis pathway of PET powder and complete the hydrolysis of PET powder to the final product terephthalic acid(TPA),this article also links the hydrolase MHETase of PET hydrolysate MHET to FAST-PETase protein,and anchors the surface of the anchor protein through cell surface display technology.pGSA-FAST-PETase-g4s-MHETase was constructed,and the N-terminus and C-terminus of the recombinant protein were exchanged to investigate the difference between the two and whether the test could directly hydrolyze PET powder to generate the final product TPA.The specific research content and results are as follows:(1)The gene of FAST-PETase was obtained by whole gene synthesis technology,the optimal surface display protein(p ET30a-pGSA)was selected,and the pGSA-FAST-PETase recombinant protein was constructed with t4 ligase and was used in E.Heterologous expression is implemented in coli BL21(DE3).Enzymatic properties showed that pGSA-FAST-PETase was used as the substrate with pGSA-FAST-PETaseat an optimal reaction temperature of 50 ℃and an optimal p H of 8.5,which proved that pGSA-FAST-PETase was a moderate-high temperature resistant alkali enzyme.In addition,pGSA-FAST-PETase has good tolerance to most common metal ions,and10 m M Na+,K+,Ca2+ ions have a certain degree of activation effect on pGSA-FAST-PETase.(2)pGSA-FAST-PETase was used to hydrolyze PET powder of different particle sizes for 24 h,and it was found that pGSA-FAST-PETase had the worst hydrolysis effect on pet powder with 50 mesh size,and the weight loss rate of powder was only 34%,but it increased with the decrease of particle size,and the hydrolysis effect of PET powder with 2000 mesh size was the best,and the weight loss rate of powder could reach 68%.pGSA-FAST-PETase hydrolysis of PET powders with different crystallinity will also have different degrees of hydrolysis.Through the hydrolysis of PET plastic bottles,it was found that the crystallinity of the bottle mouth was as low as 1.2%,while the crystallinity of the bottle body part was very high at 32.6%.In the part of the bottle mouth,the hydrolysis rate reached 52.9%,but the partial hydrolysis rate of the bottle body was only 10.2%,indicating that the hydrolysis efficiency of PET will decrease with the increase of crystallinity.The hydrolyzed 2000 mesh size PET powder was detected by HPLC,the products were TPA and MHET,and 65.4% of the PET powder was hydrolyzed after 24 h of reaction.(3)MHETase is a hydrolase of MHET,the main product of hydrolyzed PET,and the pGSA-MHETase recombinant protein is constructed by cell surface display technology,and the optimal reaction temperature of the recombinant protein is 30 ℃and the optimal p H value is 9.MHETase and FAST-PETase were linked through g4 s flexible peptides to cons truct two recombinant proteins,pGSA-FAST-PETase-g4s-MHETase and pGSA-MHETase-g4sFAST-PETase.Compared with FAST-PETase at the N-terminus of the recombinant protein,t he recombinant protein had a better hydrolysis effect at the C terminus,and the hydrolysis rate of PET was increased by 3.8%,but its MHET production increased by 17.1%,and t he production of TPA decreased by 5.6%.At the same time,during the hydrolysis of PET powder,a total of 410 μmol of MHET and 670 μmol of TPA were accumulated at 24 h.Compared to pGSA-FAST-PETase,TPA generation was increased by 294.1%,while MHET production was reduced by 51.7%.The above studies show that the efficient hydrolysis of PET plastics by constructing PET hydrolyzed proteins onto bacterial cell membranes through surface display technology is an effective means of reducing environmental pollution and reusing waste with great potential.At the same time,with the development of modern computers,computer-aided protein structure optimization is an effective supplement to traditional high-efficiency enzyme mining,but there is still great room for improvement in the comparison and utilization of high-efficiency enzymes.The results of this paper can provide some theoretical support and data support for the green pollution reduction and protein application of PET plastics.