环氧基扩链剂对3-羟基丁酸和4-羟基丁酸共聚物的改性研究

将巴斯夫代号为ADR的系列扩链剂用于3-羟基丁酸和4-羟基丁酸共聚物[P(3HB-co-4HB)]纺丝成型,分析了ADR的扩链机理,考察了P(3HB-co-4HB)和P(3HB-co-4HB)/ADR共混体系的流动性、热性稳定性、结晶性和纤维的力学性能。研究发现,以ADR作为扩链剂可有效提高熔体强度,改善纤维的热稳定性,在155℃ ADR添加量为0.5%时,熔体具有最大的剪切强度、拉伸强度和最佳的耐热性。ADR对P(3HB-co-4HB)的结晶结构影响不大,但会导致结晶速度降低和最大结晶温度提高,加入ADR的P(3HB-co-4HB)纤维的力学性能有明显改善。     

聚(3-羟基丁酸酯-4-羟基丁酸酯)/聚乳酸纤维的制备及其性能

聚(3-羟基丁酸酯-4-羟基丁酸酯)(P34HB)是极具前景的生物基可降解材料,但结晶慢且加工黏度大等因素使其难以直接熔纺制备纤维。左旋聚乳酸(PLLA)与P34HB化学结构相似,加工温度接近,与P34HB具有协同增强作用。采用熔融纺丝法制备P34HB含量为30%的P34HB/PLLA纤维,研究纤维的力学性能、热稳定性能以及结晶性能。结果发现,2 000 m/min的卷绕速率下制备的P34HB/PLLA预牵伸纤维(POY)经1.40、1.75倍牵伸和定形后得到的全牵伸纤维(FDY-1和FDY-2)的断裂强度分别为1.66、2.29 cN/dtex,断裂伸长率分别为33.3%、28.8%;差示扫描量热仪(DSC)测试结果显示,POY、FDY-1、FDY-2结晶度分别为62.51%、62.05%、61.36%;XRD衍射仪测试结果也表明POY的结晶度与FDY结晶度相近,这表明在POY成形过程中,纤维中的结晶已经接近完成,P34HB提高了PLLA结晶效率。     

P(3HB-co-4HB)生物降解性能的研究

本实验对5种4HB摩尔含量的P(3HB-co-4HB)在含有脂肪酶的磷酸缓冲液中的生物降解性能进行了研究,分析了降解机理和降解影响因素。通过质量变化、分子量变化、膜表面形态表征了其生物降解性能。结果表明,4HB含量对脂肪酶降解过程的影响较小;酶解过程中5种P(3HB-co-4HB)质量损失速率和分子量降低程度相差不大;P(3HB-co-15%4HB)具有较低的结晶度和较好的表面粗糙度,因此降解速率最快。SEM图像表明脂肪酶对于P(3HBco-4HB)具有较快的降解能力,其酶降解过程是一个表面腐蚀的过程。     

P3HB4HB/Ag纳米线复合导电纤维的制备及其性能

使用多元醇法制得银纳米线(Ag NWs),并将其与弹性体聚(3-羟基丁酸酯-co-4-羟基丁酸酯)(P3HB4HB)混合,利用湿法纺丝技术,成功制备出P3HB4HB/Ag NWs复合导电纤维。通过热重分析仪、扫描电子显微镜、电阻计及电子单纤强力仪,对复合纤维的表面形貌、导电性能及弹性回复性能进行测试和表征。结果表明,当复合纤维中Ag NWs含量为25.7%时,单根纤维电阻率达到106Ω·cm,满足标准状态下(20℃,65%RH)电阻率小于107Ω·cm的商业化导电纤维的要求;当Ag NWs含量为29.7%时,纤维内部孔洞消失,且纤维在预牵伸范围内具有良好的弹性回复性能。     

聚乳酸/聚(3-羟基丁酸酯-co-3-羟基戊酸酯)共混纤维的结构及其织物染色性能

聚乳酸(PLA)与聚(3-羟基丁酸酯-co-3-羟基戊酸酯)(PHBV)共混纺丝是改善聚乳酸(PLA)纤维的耐热性和柔韧性的方法之一。为了揭示共混纤维结构对其织物染色性能的影响规律,借助热重分析仪、差示扫描量热仪、X射线衍射仪等手段分别对共混纤维和PLA纤维的微结构和热性能进行分析。采用高、中、低温3类分散染料,对2类纤维织物的染色升温速率曲线、提升性和各项染色牢度等性能进行了比较。结果表明：共混纤维中PLA与PHBV两相分离,PLA相具有与PLA纤维相似的晶型结构,其结晶度较高,而PHBV相中形成较低的结晶;与PLA纤维相比,共混纤维的熔点较高,玻璃化温度稍低,因此其能在较低的温度下达到上染平衡;在相同的染色条件下,共混纤维织物的表观染色深度几乎是PLA织物的2倍;2类纤维织物的耐皂洗色牢度性能均不够理想。     

产聚β-羟基丁酸酯(PHB)菌种的筛选及16SrDNA鉴定

采用尼罗蓝荧光法结合苏丹黑染色法从污水中初筛获得产聚-β-羟基丁酸酯(PHB)的菌种,超声波破壁处理后采用氯仿法抽提PHB,最终通过气相色谱和紫外可见分光光度计确定了一株高产PHB菌株PK3,PHB产量为0.732g/L。经形态观察、16SrDNA序列分析,初步鉴定PK3为假单胞菌属(Pseudomonas koreensis)。     

应用电子束辐射技术的抗菌改性聚酯纳米纤维膜

为制备一种高效抗菌生物材料,以生物可降解材料聚(3-羟基丁酸酯-co-4-羟基丁酸酯)(P(3HB-4HB))和聚己二酸/对苯二甲酸丁二酯(PBAT)为基材,合成了一种新型含有季铵基团的卤胺抗菌剂单体;采用静电纺丝技术制备出P(3HB-4HB)/PBAT纳米纤维膜;利用电子束辐射技术将合成的单体接枝共聚到纳米纤维膜,最后经次氯酸钠氯化得到抗菌纤维膜。探讨了P(3HB-4HB)、PBAT组成对纤维膜表面形貌的影响,以及辐射量、单体浓度对纤维膜含氯量的影响,同时分析了抗菌纤维膜的耐紫外稳定性、储存稳定性。结果表明:P(3HB-4HB)/PBAT抗菌纳米纤维膜在5 min内即可将金黄色葡萄球菌和大肠杆菌全部杀死,显示出优异的抗菌性能,实现了卤胺抗菌剂和化学惰性材料的共价键合作用,有望应用于食品包装、生物医学等领域。     

聚乳酸活性抗菌薄膜的性能及其对樱桃保鲜效果的影响

以‘美早’大樱桃为试材,聚乳酸(polylactic acid,PLA)/聚3-羟基丁酸酯-co-4-羟基丁酸酯(poly-3-hydroxybut-yrate-co-4-hydroxybutyrate,P34HB)为基材,硅藻土附载薄荷精油为缓释型抗菌剂,采用流延成膜工艺,制备PLA/P34HB、薄荷精油-PLA/P34HB、薄荷精油/硅藻土-PLA/P34HB 3种活性抗菌膜,对活性抗菌膜性能进行测定,并通过对樱桃在(4±1)℃下贮藏期间保鲜袋内顶空气体组成、质量损失率等理化指标的测定以及低场核磁共振(low field-nuclear magnetic resonance,LF-NMR)分析樱桃内部水分状态的变化,研究不同薄膜和对照(不加薄膜)对樱桃保鲜效果的影响。结果表明:具有硅藻土附载薄荷精油的薄膜相较于PLA/P34HB薄膜氧气透过率从250.37 cm³/(m²·d·0.1 MPa)增加到290.45 cm³/(m²·d·0.1 MPa),有利于维持樱桃品质。LF-NMR结果表明含有硅...     

聚乳酸/聚3-羟基丁酸-戊酸酯共混纤维及其雪尼尔纱的染色动力学

为进一步了解生物可降解聚乳酸/聚3-羟基丁酸-戊酸酯(PLA/PHBV)共混纤维及其雪尼尔纱的染色性能，提高对含生物可降解聚酯纤维雪尼尔纱染色技术的可控性，研究了PLA/PHBV纤维及PET/(PLA/PHBV)雪尼尔纱的染色性能，探究了C. I.分散红60在雪尼尔纱及其原材料纤维上的染色动力学，并对各纤维的生物可降解性能进行了研究。结果表明：C. I.分散红60在PLA/PHBV纤维及PET/(PLA/PHBV)雪尼尔纱上的上染率随着温度的升高而明显提升，但为兼顾纤维的力学性能，染色温度不宜超过110℃;C. I.分散红60在PET纤维、PLA/PHBV纤维及雪尼尔纱3种材料上的吸附均符合准二级动力学方程，其在PLA/PHBV纤维上的染色平衡吸附量最高，在PET纤维上的最低，并且随着染色温度升高，C. I.分散红60在3种材料上的染色速率加快，半染时间缩短，扩散系数增大；各纤维的降解效率均随土埋时间的延长而提高，其中PLA/PHBV纤维的降解性能最好，PLA纤维次之，且PLA或PLA/PHBV生物可降解聚酯纤维的存在，可加快PET纤维的降解速率。     

聚羟基脂肪酸酯/海藻酸钠纳米纤维的制备及其性能

针对聚羟基脂肪酸酯(P(3HB-co-4HB))和海藻酸钠(SA)常规情况下难共溶的问题,以P(3HB-co-4HB)为原料,SA为改性剂,三氯甲烷/水为溶剂,烷基糖苷(APG)为乳化剂,通过共混利用静电纺丝法制备了P(3HB-co-4HB)/SA纳米纤维膜。借助红外光谱仪、差示扫描量热仪、扫描电子显微镜表征P(3HB-co-4HB)/SA静电纺纳米纤维的分子间作用力、热性能和形貌;利用细胞毒性和细胞共培养测试表征了P(3HB-co-4HB)/SA纳米纤维的生物相容性。结果表明:P(3HB-co-4HB)/SA复合材料的玻璃化转变温度发生改变,当添加SA质量分数为6%时,静电纺纳米纤维具有均一的形貌,纳米纤维的平均直径为500 nm,孔隙率为74%,细胞毒性等级为0级,P(3HB-co-4HB)和SA具有良好的生物相容性。     

Production and characterization of biodegradable terpolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) by Alcaligenes sp. A-04

The production of the terpolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate), P(3HB-co-3HV-co-4HB), by Alcaligenes sp. A-04 was investigated to determine the superior biodegradable polymer properties over those of poly(3-hydroxybutyrate), P(3HB), and its copolymers. The highest terpolymer content of 68% (w/w) was produced by Alcaligenes sp. A-04 at 60 h by shake-flask cultivation. The terpolymer with 93 mol% 4HB mole fraction units was produced when the cultivation time was extended to 96 h. Moreover, it was found that Alcaligenes sp. A-04 could utilize 1,4-butanediol for the synthesis of 3HB and 4HB monomers as well as the sodium salt of 4-hydroxybutyrate. The terpolymer content was 30% (w/w) and the composition was P(33%3HB-co-16%3HV-co-51%4HB). Next, terpolymers with 4HB mole fraction units ranging from 50 to 90 mol% were produced by varying the medium composition and cultivation time. The thermal and mechanical properties of the resulting terpolymers were different from those of the copolymers with a similar mole fraction of monomer units. The terpolymer P(4%3HB-co-3%3HV-co-93%4HB) showed an elongation of 430%, a toughness of 33 MPa, and Young's modulus of 127 MPa similar to those of low-density polyethylene. The terpolymer P(11%3HB-co-34%3HV-co-55%4HB) showed Young's Modulus of 618 MPa similar to that of polypropylene.     

Enzymatic synthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) with CoA recycling using polyhydroxyalkanoate synthase and acyl-CoA synthetase

We succeeded in developing a novel method for in vitro poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3 HB-co-4 HB)] synthesis with CoA recycling using polyhydroxyalkanoate synthase and an acyl-CoA synthetase. Using this method, the monomer compositions in P(3 HB-co-4 HB)s could be controlled strictly by the ratios of the monomers in the reaction mixtures.     

Regulating the molar fraction of 4-hydroxybutyrate in poly(3-hydroxybutyrate-4-hydroxybutyrate) biosynthesis by Ralstonia eutropha using propionate as a stimulator

The regulation of the molar fraction of 4-hydroxybutyrate (4-HB) in the poly(3-hydroxybutyrate-4-hydroxybutyrate) [P(3HB-4HB)] biosynthesis by Ralstonia eutropha (formerly Alcaligenes eutrophus) was attempted by the supplemental addition of propionate. The molar fraction of 4-HB in P(3HB-4HB) was increased significantly from 12.3 to 51.8 mol% by the addition of a small amount of propionate along with gamma-butyrolactone commonly used as a precursor for the biosynthesis of P(3HB-4HB). The mechanism of regulation by propionate was investigated by measuring the variation of enzyme activities related to the biosynthesis of P(3HB-4HB) and the level of intermediate metabolite acetyl-CoA. PHB synthase activity was induced significantly by propionate, and the acetyl-CoA concentration also increased significantly due to the additional supply of propionate. The overflowing acetyl-CoA seems to cause an inhibitory effect on the ketolysis reaction catalysing the lysis of 4-hydroxybutyryl-CoA to two molecules of acetyl-CoA; consequently, the 4-HB fraction available for polymerization increased. Accordingly, the molar fraction of 4-HB in P(3HB-4HB) biosynthesis seems to be regulated by both an increased 4-HB fraction and an activated PHB synthase due to the supplemental addition of propionate as a stimulator.     

Fermentative production of (R)-(-)-3-hydroxybutyrate using 3-hydroxybutyrate dehydrogenase null mutant of Ralstonia eutropha and recombinant Escherichia coli

Two systems, one using an (R)-(-)-3-hydroxybutyrate dehydrogenase (BDH) null mutant of Ralstonia eutropha and the other using a recombinant Escherichia coli strain containing a synthetic poly[(R)-(-)-3-hydroxybutyrate] (PHB) operon and an extracellular PHB depolymerase gene, were used for the fermentative production of (R)-(-)-3-hydroxybutyrate (3HB). The concentration of 3HB in the culture supernatant of the mutant R. eutropha system reached about 30 mM after 5 d under anaerobic conditions, although it was about 4-10 mM under aerobic conditions. On the other hand, the 3HB concentration in the culture supernatant of the recombinant E. coli system reached about 70 mM after 4 d, indicating that about 70% of the glucose added was converted to 3HB.     

Characterization of two 3-hydroxybutyrate dehydrogenases in poly(3-hydroxybutyrate)-degradable bacterium, Ralstonia pickettii T1

Two D-(-)-3-hydroxybutyrate (3HB) dehydrogenases, BDH1 and BDH2, were isolated and purified from a poly(3-hydroxybutyrate) (PHB)-degradable bacterium, Ralstonia pickettii T1. BDH1 activity increased in R. pickettii T1 cells grown on several organic acids as a carbon source but not on 3HB, whereas BDH2 activity markedly increased in the same cells grown on 3HB or PHB. To examine their biochemical properties, bdh1 and bdh2 were cloned and overexpressed in Escherichia coli, and their purified products were characterized. The kinetic parameters indicate that BDH1 is more suitable for converting acetoacetate to 3HB than BDH2, whereas BDH2 is more efficient for the reverse reaction than BDH1. Thus, R. pickettii T1 contains two BDHs with different biochemical properties and physiological roles: BDH1 for cell growth on organic acids other than 3HB and BDH2 for cell growth on 3HB or PHB.     

Improvement of poly(3-hydroxybutyrate) [P(3HB)] production in Corynebacterium glutamicum by codon optimization, point mutation and gene dosage of P(3HB) biosynthetic genes

In our previous study, a system for producing poly(3-hydroxybutyrate) [P(3HB)] was established by introducing a polyhydroxyalkanoate (PHA) biosynthetic gene operon (phaCAB Re) derived from Ralstonia eutropha into Corynebacterium glutamicum. In this study, two experimental strategies have been applied to improve P(3HB) production in recombinant C. glutamicum. One is a codon optimization of the N-terminal-coding region of the PHA synthase (PhaC Re) gene focusing on the codon usage preference for the translation system of C. glutamicum. The other is the replacement of wild-type phaC Re with a modified gene encoding a mutation of Gly4Asp (G4D), which enhanced the production of PhaC Re and P(3HB) in Escherichia coli. The introduction of these engineered PHA synthase genes into C. glutamicum enhanced the production of PhaC(Re) and P(3HB). Interestingly, we found that these gene modifications also caused increases in the concentration of the translation products of the genes encoding monomer-supplying enzymes, beta-ketothiolase (PhaA Re) and acetoacetyl-CoA reductase (PhaB Re). This finding prompted us to carry out a gene dosage of phaAB Re for a double plasmid system, and the highest production (52.5 wt%) of P(3HB) was finally achieved by combining the gene dosage of phaAB Re with codon optimization. The molecular weight of P(3HB) was also increased by approximately 2-fold, as was P(3HB) content. Microscopic observation revealed that the volume of the cells accumulating P(3HB) was increased by more than 4-fold compared with the non-P(3HB)-accumulating cells without filamentous morphologenesis observed in E. coli.     

Production system for biodegradable polyester polyhydroxybutyrate by Corynebacterium glutamicum

A biosynthetic pathway for poly(3-hydroxybutyrate) [P(3HB)] production by Corynebacterium glutamicum was developed by introducing the phbCAB operon derived from Ralstonia eutropha. P(3HB) synthase activity was detected in this recombinant C. glutamicum carrying a cell surface protein gene promoter. Intracellular P(3HB) was microscopically observed as inclusion granules and its content was calculated to be 22.5% (w/w) with a number average molecular weight of 2.1x10(5) and a polydispersity of 1.63.     

Poly[(R)-3-hydroxybutyrate] formation in Escherichia coli from glucose through an enoyl-CoA hydratase-mediated pathway

In this study, a new metabolic pathway for the synthesis of poly[(R)-3-hydroxybutyrate] [P(3HB)] was constructed in a recombinant Escherichia coli strain that utilized forward and reverse reactions catalyzed by two substrate-specific enoyl-CoA hydratases, R-hydratase (PhaJ) and S-hydratase (FadB), to epimerize (S)-3HB-CoA to (R)-3HB-CoA via a crotonyl-CoA intermediate. The R-hydratase gene (phaJ(Ac)) from Aeromonas caviae was coexpressed with the PHA synthase gene (phaC(Re)) and 3-ketothiolase gene (phaA(Re)) from Ralstonia eutropha in fadR mutant E. coli strains (CAG18497 and LS5218), which had constitutive levels of the beta-oxidation multienzyme FadB(Ec). When grown on glucose as the sole carbon source, the cells accumulated P(3HB) up to an amount 6.5 wt% of the dry cell weight, whereas the control cells without phaJ(Ac) or fadR mutation accumulated significantly smaller amounts of P(3HB). These results suggest that PhaJ(Ac) and FadB(Ec) played an important role in supplying monomers for P(3HB) synthesis in the pathway. Furthermore, by using this pathway, a P(3HB)-concentration-dependent fluorescent staining screening technique was developed to rapidly identify cells that possess active R-hydratase.