Efficient Expression Of Plastic Degrading Enzyme(PETase)in Bacillus Subtilis And Improvement Of Its Enzymatic Properties

The widespread use of polyethylene terephthalate plastic?PET?has caused serious pollution to the ecological environment,and the secondary pollution caused by incineration and chemical degradation is also severe.Therefore,more gentle biodegradation offers the possibility of PET recycling.It is reported that PETase,a plastic degrading enzyme derived from Ideonella sakaiensis,exhibits higher PET hydrolysis activity than other enzymes.Bacillus subtilis is a recognized biosafety strain with strong secretion ability and a good heterologous gene expression host.Promoters are important genomic regulatory elements that directly affect gene expression levels,and signal peptides determine the protein's exocrine capacity.Therefore,the successful and efficient expression of foreign genes requires not only the selection of a suitable host strain,but also the appropriate promoter and signal peptide.In this study,we used Bacillus subtilis WB600 for exocrine expression of PETase,and selected 5signal peptides and 2 promoters to mediate the extracellular secretion of PETase,and used the natural signal peptide SPPETase as a control.Through orthogonal design,7 engineering bacteria were constructed,respectively:WB600-P43-SP_AprE,WB600-P43-SP_apr,WB600-P43-SP_BprA,WB600-P43-SP_SacC,WB600-P43-SP_amy,WB600-P43-SP_PETase and WB600-Pylb-SPamy.Since PETase is easily degraded quickly at 40°C,the culture temperature of the engineering bacteria was screened,and it was found that the bacterial growth was fast at 28°C and the enzyme activity hardly decreased within 48 hours.Comparison of the signal peptides revealed that the amy signal peptide had an exocrine effect4.5 times greater than that of the natural signal peptide.The other four signal peptides,apr,SacC,BprA,and AprE,all had more exocrine effects than natural signal peptides.Then the comparison of P43 and Pylb promoters on the activity of exo-expressed PETase showed that the effect of weak promoter P43 was 15%higher than that of strong promoter Pylb,which may be due to the weak promoter transcription and protein folding Provides more time.Therefore,P43 and amy are the optimal combination of PETase expressed exogenously in Bacillus subtilis.Further experimental results show that P43 and amy-mediated secretion of PETase can degrade 80%of BHET?PET monomer?within 2 hours,and when it interacts with PET membrane,it can be clearly observed under scanning electron microscope within 36hours The PET film is corroded.For the PETase exogenously expressed in Bacillus subtilis,this is also the first time that a signal peptide with a more significant exocytosis effect than the natural signal peptide has been discovered.However,because the glass transition temperature of PET is higher than 70°C,the process of degrading PET needs to be carried out at high temperature.PETase is easily degraded at 40°C,and its low thermal stability constitutes a bottleneck for the actual enzymatic degradation of PET.Therefore,it is particularly important to improve the thermal stability of PETase.In this study,the model was built by machine learning,and PETase was used as the research object,the mutations with increased?T_m were counted,and the conservative regions were removed.Then,19 mutants were designed and the structure of PETase was modified.Finally,9 mutants whose T_m value increased by more than 0.5°C were obtained.Among them,the T_m value of S136Q mutant increased by 1.56°C,the optimal temperature increased by 5°C,and the enzyme activity was still as high as 79.1%after incubation at 50°C for 60 min.Compared with the wild type,the stability is improved by 1.5times.In this study,the screening of signal peptides and promoters significantly increased the exogenous expression of PETase in Bacillus subtilis,and provided an effective means for the industrial application of PETase enzyme preparations.In addition,based on the semi-rational design,the thermal stability of PETase and other enzymatic properties have been improved,laying a theoretical foundation for the industrial application of PETase.