The Construction And Characterization Of Hydrophobic Peptide-cutinase Fusion Protein And Its Application In PET Degradation

Polyethyleneterephthalate（PET）has been widely used dueto its excellent properties.With thedevelopment of global economy,theproduction of PET has increased dramatically,and its total amount of plastic wasteis increasing every year.Thetreatment methods of PET wastemainly includelandfill,incineration and biodegradation.Landfilling and incineration can causesecondary pollution,so biodegradation has becomea research hotspot dueto its environmentally friendly property.It is mainly enzymatic degradation.PET hydrolases aremainly cutinase,which is a multi-functional hydrolasethat can degradePET ester bonds.However,thehigh hydrophobicity and compact structureof PET limit thebinding and degradation by cutinase.Relevant studies had shown that hydrophobic peptide-cutinasefusion protein modifies PET fibers,and theeffect was significantly improved.Therefore,this paper fused cutinasewith a variety of short hydrophobic peptides to increasethedegradation rateof PET.Themain research results wereas follows:（1）Screening of hydrophobic peptides.Fivehydrophobic short peptides（BaCBM2,DSI,TA2,HFB4 and HFB7）wereselected to fusewith theN terminal of theenhanced green fluorescent protein.Thesurfacefluorescencesignal of PET membranetreated with BaCBM2-eGFP and DSI-eGFP was stronger,indicating that BaCBM2 and DSI had stronger adsorption ability for PET.Theabsorption of hydrophobic peptidewas analyzed.Trp9,Trp44 and their surrounding polar amino acids of BaCBM2 aremorelikely to becloseto thesurfaceof PET and form hydrophobic accumulation with PET benzenering.However,dueto thehigh proportion of hydrophobic non-polar amino acids in amino acid sequence,DSI may enhancethesurfacebinding ability of hydrophobic PET To further explain thepotential reason for thestrong adsorption of BaCBM2 and DSI to PET.（2）Optimization of functional characterization of DSI-cutinasedegradation of PET.Thefusion proteins BaCBM2-Tfuc and DSI-Tfuc wereconstructed by combining BaCBM2,DSI and Thermobifida fusca cutinase（Tfuc）.Theoptimum p H was 8.0 and theoptimum temperaturewas 80℃.Thehalf-lifeof BaCBM2-Tfuc and DSI-Tfuc at 60℃were30 h and 60 h,respectively.Thedegradation rates of BaCBM2-Tfuc and DSI-Tfuc to PET were2.8 times and3.7 times that of Tfuc,respectively.At thesametime,theenzymatically treated PET film was observed by scanning electron microscope,and thedegradation degreewas DSI-Tfuc>BaCBM2-Tfuc>Tfuc.It was further demonstrated that DSI could improvetheadsorption rateof Tfuc on PET film,thus improving thedegradation efficiency of PET.Therefore,thehydrophobic peptideof DSI was selected as thefollow-up research object.（3）Thefunction optimization of DSI-cutinaseenzymatic degradation of PET.DSI was fused with strong heat resistanceof cutinase,including Tfuc mutant Tfuc2（D204C/E253C）and Leaf-branch compost cutinase（LCC）mutant ICCG（F243I/D238C/S283C/Y127G）to obtain thefusion proteins DSI-Tfuc2 and DSI-ICCG.Theoptimum p H was 8.0.Theoptimal temperatureof DSI-Tfuc2 was 80℃.However,theoptimal temperatureof DSI-ICCG did not detect theinflection point（30℃-100℃）.Compared with thewild type,thethermal stability of DSI-Tfuc2 and DSI-ICCG was improved.Theanalysis of kinetic parameters found that theK_mvalueof thefusion protein decreased,and both thek_cat and catalytic efficiency(k_cat/K_m)increased.Thedegradation rateof DSI-Tfuc2（58.8%）was 32.5 times higher than that of Tfuc2at 70℃.Thedegradation rates of ICCG and DSI-ICCG both reached morethan 90.0%at 60℃and 70℃.（4）Theexpression optimization of thefusion protein.Different culturemedia（LB,TB,ZYM）and expression vectors（pET-20b（+）and pET-24a（+））had no significant differencein theexpression of thefusion protein;compared with DSI-（GGGGS）₃-ICCG,S1v1（ANANARAR）₂was fused to theN terminus of DSI-ICCG,theintracellular enzymeactivity was increased by2.1 times;theenzymeactivity of direct fusion between DSI and ICCG was 2-4 times that of DSI-（GGGGS）_n-ICCG（n≤3）;in addition,thefusion protein was expressed in P.pastoris.Compared with Escherichia coli,therewas no significant increasein protein expression,but theprotein band was single,which was conduciveto later purification applications.