【作者】 拉莎米（Lakshmi Tripathi）；

【导师】 陈国强；

【作者基本信息】 清华大学 ， 生物学， 2013， 博士

【摘要】 聚羟基脂肪酸酯（PHA）是很多微生物都可合成的具有生物可降解性的高分子聚酯。为满足对生物塑料日益增长的需求，具有多样化学结构和独特性质的PHA越来越受重视。利用微生物法生产嵌段共聚物（block copolymer PHA）整合了聚合物微结构的优势，从而获得一些其它PHA（包括均聚物、混聚物、随机共聚物）所没有的、新型实用的性质。本课题合成了以下三种嵌段共聚物：（1）聚3-羟基丁酸酯-block-聚3-羟基己酸酯（P3HB-b-P3HHx）；（2）聚3-羟基丙酸酯-block-聚4-羟基丁酸酯（P3HP-b-P4HB）；（3）聚3-羟基己酸酯-block-（聚3-羟基十酸酯-co-聚3-羟基十二酸酯）［P3HHx-b-P(3HD-co-3HDD)］。同时通过1H NMR，13CNMR，HMBC NMR，DSC，GPC和机械性能测试研究这些嵌段共聚物的热学和机械性质。首先，我们用-氧化弱化的恶臭假单胞菌Pseudomonas putida合成了聚3-羟基丁酸酯-block-聚3-羟基己酸酯P3HB-b-P3HHx。聚3-羟基丁酸酯是短链PHA，而聚3-羟基己酸酯是中长链PHA，两者通过共价连接形成二聚嵌段聚合物。P3HB-b-P3HHx比随机共聚物P(3HB-co-P3HHx)具有更优良的机械性质。利用重组大肠杆菌将柔软高弹性的聚4-羟基丁酸酯（P4HB）和强刚性的聚3-羟基丙酸酯（P3HP）连接形成嵌段聚合物，以获得性能优异的材料。本研究获得两种嵌段聚合物P3HP-b-29%P4HB和P3HP-b-37%P4HB，比随机共聚物P(3HP-co-4HB)具有更优的性质。与均聚物P3HP和P4HB相比，嵌段微结构的引入降低了熔融温度。此外，嵌段聚合物比之前报道的组分相近的随机共聚物具有更高的杨氏模量（Young’s modulus）、屈服强度（Yield strengths）和拉伸强度（Tension strengths）。最后，以基因组精简的恶臭假单胞菌为工程菌株，通过连续的添加相关脂肪酸碳源，得到中长链PHA嵌段聚合物P3HHx-b-P(3HD-co-3HDD)，其中P3HHx和P(3HD-co-3HDD)分别含有49mol%和51mol%。热力学性质分析表明，该嵌段共聚物同时获得了3HHx和3HD的无定型和3HDD的高结晶，并且比中长链均聚物和随机共聚物PHA具有更高的力学强度。这项研究的目的主要是利用微生物法生产嵌段共聚物，不仅丰富了PHA的结构组成，在一定程度也提高了PHA的各项理化性质。微生物法生产嵌段共聚物通过共价连接两种甚至更多种的均聚物从而增强了材料的特性，通过控制聚合物单体的组成可以得到具有理想性质的生物材料。嵌段聚合开辟了一种新的聚羟基脂肪酸酯的聚合形成方式，并表现出优越的性能。在我们的研究工作中，恶臭假单胞菌KT2442作为研究平台，可以合成出多种新型生物聚合物，尤其以聚羟基脂肪酸酯的嵌段共聚物和无序共聚物为主。这项工作可以生产出具有可持续发展的环境友好型生物聚合物并用来替代石油基聚合物。更多还原

【Abstract】 Polyhydroxyalkanoates (PHA) are fully biodegradable biopolymers produced byseveral microorganisms from renewable substrates. To meet the current growingrequirements of bioplastics, PHA biopolymers with diverse chemical structures andunique properties are required. Microbial block copolymerization offers the advantageof incorporating polymer microstructures that achieve some novel properties not yetseen in PHA homopolymers, blend polymer and random copolymers. In the presentstudy three types of block copolymers were biosynthesized such aspoly(3-hydroxybutyrate)-block-poly(3-hydroxyhexanoate)[P3HB-b-P3HHx],poly(3-hydroxypropionate)-block-poly(4-hydroxybutyrate)[P3HP-b-P4HB],poly(3-hydroxyhexanoate)-block-poly(3-hydroxydecanoate-co-3-hydroxydodecanoate)[P3HHx-b-P(3HD-co-HDD)]. The block copolymers were characterized by1H NMR,13C NMR, HMBC NMR, DSC, GPC and mechanical property analysis.Diblock copolymers comprising of poly-3-hydroxybutyrate (P3HB) as theshort-chain-length (SCL) PHA block covalently bonded with poly-3-hydroxyhexanoate(P3HHx) as the medium-chain-length (MCL) PHA block were for the first timesuccessfully produced in-oxidation weakened Pseudomonas putida KT2442.Differential scanning calorimetry (DSC) showed that the block copolymer had two glasstransition temperatures (Tg), one melting temperature (Tm) and one cool crystallizationtemperature (Tc), it had superior mechanical properties over random copolymerP(3HB-co-3HHx).Block copolymers consisting of elastic P4HB block with a strong and tough P3HPblock were biosynthesized to gain unique and excellent material properties, two blockcopolymers were formed from this study, including the P3HP-b-29mol%P4HB andP3HP-b-37mol%P4HB, they showed superior properties over random copolymersP(3HP-co-4HB) with similar monomer ratios. The block copolymers had two glasstransition temperatures (Tg) and two melting temperatures (Tm). In comparison to thehomopolymers P3HP and P4HB, incorporation of block microstructure resulted in thelowering of Tm, block copolymers were revealed with higher Young’s modulus, yield strengths and tension strengths, much better than the previously reported randomcopolymers of similar compositions.Medium chain length PHA (MCL PHA) diblock copolymerP3HHx-b-P(3HD-co-3HDD) consisting of a49mol%poly-3-hydroxyhexanoate (3HHx)block with another block consisting of51mol%copolymer of3-hydroxydecanoate(3HD) and3-hydroxydodecanoate (3HDD) was produced from another β-oxidationweakened strain Pseudomonas putida KT2442during the sequential addition of relatedfatty acid substrates. Thermo-mechanical properties of the block copolymer showed thatblock microstructure gained the amorphous nature of3HHx and3HD and attained thecrystallinity of3HDD, thus higher mechanical strength was realized in contrast tomedium chain length MCL PHA homopolymers and random copolymer. PHAproduction platform consisting of P. putida derivatives was also utilized for theproduction of copolyesters of P(3HB-co-3HHx) and P(3HHx-co-3HD) with a widerrange of alterable monomer contents and compositions.The purpose of this research is to microbially produce block copolymers to diversifyPHA microstructures and increase their material properties. Microbial production ofblock copolymers can covalently link together two or more homopolymers and gainnovel material properties. Thus, desired material properties can be gained based on thePHA monomer composition. In this study, Pseudomonas putida KT2442was developedas a suitable platform for the production of various new biopolymers, especiallypolyhydroxyalkanoates (PHA) block copolymers, and random copolymers. This studycould help to produce sustainable bio based polymers in order to replace petroleumbased polymers.更多还原