【作者】 张颖；

【导师】 田亚平；

【作者基本信息】 江南大学 ， 发酵工程， 2021， 硕士

【摘要】 聚对苯二甲酸乙二醇酯（PET）因具有优良的特性,被广泛应用,随着全球经济的发展,PET生产量大幅提高,其废弃总量也逐年增加。废弃PET处理方法主要包括填埋、焚烧及生物降解。填埋和焚烧均会造成二次污染,而具有环境友好特性的生物降解逐渐成为研究热点,主要是酶法降解,PET水解酶类以角质酶为主,其是一种多功能水解酶可以降解PET的酯键。然而PET的高度疏水性以及紧密的结构限制了角质酶对其的结合与降解。相关研究表明,疏水短肽-角质酶融合蛋白对PET纤维进行改性,效果提升显著。因此本论文将角质酶与多种疏水短肽构建融合蛋白,实现PET的高效降解。主要研究结果如下:（1）疏水短肽的筛选。选择五种疏水短肽（BaCBM2、DSI、TA2、HFB4和HFB7）,与增强型绿色荧光蛋白（eGFP）基因N端融合。PET膜经BaCBM2-eGFP、DSI-eGFP处理后,表面荧光信号较强,表明BaCBM2、DSI对PET具有更强的吸附性。对疏水短肽吸附性进行分析,BaCBM2的氨基酸序列中Trp9、Trp44及其周围极性氨基酸,更易靠近PET表面,与PET苯环形成疏水堆积作用。而DSI由于氨基酸序列中疏水非极性氨基酸占比高,可能导致对疏水PET表面结合能力增强,解释了BaCBM2、DSI对PET强吸附性的潜在原因。（2）疏水短肽-角质酶酶法降解PET的功能表征。将BaCBM2,DSI与嗜热子囊菌Thermobifida fusca来源的角质酶（Tfuc）构建融合蛋白BaCBM2-Tfuc和DSI-Tfuc,其最适p H均为8.0,最适温度均为80℃。BaCBM2-Tfuc和DSI-Tfuc在60℃下半衰期分别为30 h和60 h。BaCBM2-Tfuc和DSI-Tfuc对PET的降解率分别是Tfuc的2.8倍和3.7倍。同时通过SEM观察酶法处理后的PET膜,表面侵蚀程度DSI-Tfuc>BaCBM2-Tfuc>Tfuc,进一步说明DSI对PET膜的吸附性更强。因此选择DSI疏水短肽作为后续研究对象。（3）DSI-角质酶酶法降解PET的功能优化。将DSI与耐热性强的角质酶融合,包括Tfuc突变体Tfuc2（D204C/E253C）和Leaf-branch compost来源的角质酶LCC突变体ICCG（F243I/D238C/S283C/Y127G）,得到融合蛋白DSI-Tfuc2和DSI-ICCG,其最适p H均为8.0。DSI-Tfuc2最适温度为80℃,而DSI-ICCG最适温度未检测到拐点（30℃-100℃）。与野生型相比,DSI-Tfuc2和DSI-ICCG热稳定性均有所提高。动力学参数分析发现,融合蛋白K_m值均减小,k_cat及催化效率(k_cat/K_m)均增加。DSI-Tfuc2在70℃反应4 d的降解率（58.8%）较Tfuc2提高32.5倍。ICCG和DSI-ICCG在60℃和70℃下反应4 d的降解率均达到90.0%以上。（4）融合蛋白的表达优化。不同培养基（LB、TB、ZYM）培养和更换不同表达载体（pET-20b（+）和pET-24a（+））对融合蛋白表达量的影响无明显差别;相比较DSI-（GGGGS）₃-ICCG,S1v1（ANANARAR）₂融合至DSI-ICCG的N端,胞内酶活提高2.1倍;DSI和ICCG直接融合的酶活是DSI-（GGGGS）_n-ICCG（n≤3）的2-4倍;此外在毕赤酵母中进行融合蛋白的表达,与大肠杆菌表达相比,虽然蛋白表达量无明显提高,但是蛋白条带单一,利于后期纯化应用。更多还原

【Abstract】 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.更多还原