Recombinant Expression,molecular Modification,and Application Of Humicola Insolens Cutinase-OMP25

Stickies are pollutants generated during the recycling process of waste paper,which are easily retained on the surface of paper and equipment,affecting product quality,causing equipment damage and seriously affecting the efficiency of waste paper recycling.The control of stickieshas become an urgent problem to improve the utilization rate of waste paper.The control methods of stickies include physical,chemical and biological methods.Biological control methods are currently of great interest,which are environmentally friendly,efficient and specific,and can effectively degrade the main components of stickies such as polyvinyl acetate（PVAc）and polyethyl acrylate（PEA）.Cutinase is a multifunctional enzyme with polyester degradation ability.Meanwhile,anchor peptide is a peptide that can anchor to specific substrates.Preliminary studies have shown that the fusion of the two significantly improved the degradation of stickies by cutinase.In this study,the fusion protein of Humicola insolens cutinase（Hi C）with the anchor peptide OMP25（Hi C-OMP25）was selected for study.First,the differences in Hi C-OMP25 of expression in different Escherichia coli,substrate degradation,enzyme stability,and cell membrane permeability and cell surface hydrophobicity of the host bacteria were explored to reveal the differences when Hi C-OMP25 was expressed in different host bacteria.Secondly,through strategies of replacing ribosome binding site（RBS）,linker and co-expression of chaperone protein,the expression of fusion protein was further improved.Hi C-OMP25 was then prepared efficiently.Then,the storage stability of fermentation broth was improved by additives.Finally,the molecular modification of anchor peptide was used to further improve the degradation effect of fusion protein on stickies.The main findings are as follows:（1）The variability in the expression of Hi C-OMP25 by different host bacteria was examined.In this study,Hi C-OMP25 was expressed in E.coli BL21（DE3）and E.coli C43（DE3）strains,respectively,and p-nitrophenyl butyrate（p NPB）activity was detected in the fermentation supernatant with enzyme activities of 207.9 U·m L^-1 and 43.1 U·m L^-1,respectively.However,Hi C-OMP25 expressed in E.coli BL21（DE3）was found to be less effective in degrading PEA and less stable at 50℃than Hi C-OMP25 expressed in E.coli C43（DE3）.Meanwhile,the cell membrane permeability and cell surface hydrophobicity of E.coli BL21（DE3）strain expressing Hi C-OMP25 were significantly enhanced compared with those of E.coli BL21（DE3）strain expressing Hi C-OMP25 significantly enhanced compared to Hi C-expressing E.coli BL21（DE3）strain.It was hypothesized that Hi C-OMP25 could fold correctly in E.coli C43（DE3）,while it failed to fold completely when expressed in E.coli BL21（DE3）,leading to the difference in the degradation effect on PEA and thermal stability at50℃.（2）The effects of different RBS,linker and chaperone proteins on Hi C-OMP25 expression in E.coli C43（DE3）were investigated,and the high-density fermentation of the optimal recombinant strain was carried out,followed by optimization of Hi C-OMP25 storageadditives.The experimental results showed that the recombinant strain Hi C-OMP25 before RBS and linker replacement has higher expression.However,co-expression of Hi C-OMP25 with sulfhydryl oxidase（Erv1p）and disulfide bond isomerase（Dsb C）in E.coli C43（DE3）resulted in an extracellular enzyme activity of 135.3 U·m L^-1 in the shake flask,which was 2.14 times higher compared with the original strain.In the high-density fermentation,the highest enzyme activity for E.coli C43（DE3）/p ET-20b（+）-hic-omp25/p CDFDuet-1-erv1p-dsbc recombinant strain was 855.6 U·m L^-1 in the 3-L fermenter.It was demonstrated that the addition of 20%glycerol+3‰potassium sorbate+3‰sodium benzoate+10%PEG600 and 10%sorbitol could increase the relative residual enzyme activity of Hi C-OMP25 from 52%to 84%after 336 h at50°C.（3）Molecular modification of anchoring peptides to improve the degradation of adhesive by fusion proteins.Two mutants（M3 and M6）with improved degradation efficiency of PEA compared to Hi C-OMP25 were obtained by molecular modification of the anchoring peptide,and the relative turbidity after degradation of PEA was reduced from 22%in wild type to 13%and 17%,respectively.Based on the improved degradation of PEA by M3 and M6,the stacked mutants of M3 and M6,M7 were constructed.The optimum reaction conditions and stability of M3,M6 as well as M7 mutants were similar to those of Hi C-OMP25,with the optimum p H of 8.5,the optimum temperature of 80℃,and the half-lives of 192 h and 168 h at 30℃and50℃,respectively.M3,M6 and M7 all improved the degradation efficiency of polyvinyl acetate（PVAc）,and their degradation rate of PVAc increased from 25%in the wild type to 35%,41%and 29%,respectively.According to the degradation of M3,M6 and M7 on the actual stickies,it was found that M6 showed no significant change in degradation efficiency compared with Hi C-OMP25;M3 and M7 showed 1.06 and 0.97 times increase in turbidity change values of stickies compared with Hi C-OMP25,respectively.The results showed that mutant M3 helped to improve the degradation effect of Hi C on stickies,which provided theoretical guidance and technical support for the subsequent industrial application.