从微生物发酵液中分离提取PHB
文献类型：学位论文
| 作者 | 秦杰 |
| 学位类别 | 博士 |
| 答辩日期 | 1999-11 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 欧阳藩 |
| 关键词 | 絮凝 自然絮凝 粘度法 次氯酸钠 过氧化氢 结晶形态 溶解度 三氯甲烷 欧文氏菌 大肠肝菌 |
| 其他题名 | Separating PHB from Microorganism Broth |
| 学位专业 | 化学工艺 |
| 中文摘要 | 目前“白色污染”正日益严重地威胁着自然环境，它造成了如土地质量严重退化而使农业减产等许多问题，世界各国也为寻找对策进行了大量的研究，其中使用可降解塑料是一个最有前途、最有效的办法，PHB（聚－β－羟基丁酸）正是其中研究最早、应用前景最广泛的生物可降解材料。但是，目前由于其成本较高，与廉价的石油工业高分子材料在价格上无法竞争，影响了它的广泛使用。为了降低成本，提高生产效率，本文针对发酵法生产PHB过程中存在的，发酵液难以分离沉淀，非有机溶剂提取PHB时分子量严重降低等问题，进行了发酵液的絮凝，及温度、pH、氧化剂对提取PHB影响的研究。并对粘度法测定PHB分子量的方法进行了研究，同时，对于研究过程中发现的，加热后PHB在三氯甲烷中的溶解性变化及其原因进行了探索。首次在转基因欧文氏工程菌发酵液体系下，对絮凝剂作了筛选，并对其对最佳作用条件进行了研究。结果表明，发酵液的pH、絮凝剂浓度和絮凝温度对絮凝效果的影响较大。在酸性条件下ZTC、J－101作用效果较好；而在碱条件下ZTC、净水粉、明矾、硫酸铝钾、J－101、聚合铝、的作用效果较好，其中ZTC、J－101和净水粉絮凝效果最好。首次对欧文氏菌发酵液进行了自然絮凝的研究。研究表明，在不加任何絮凝剂的情况下，将发醇液调到酸性或碱性都会使其发生自然絮凝，而且在pH < 4的酸性条件下自然絮凝效果最好。进一步的实验说明自然絮凝的发生与Zeta电位的绝对值降低是相关联的。对粘度法测定PHB分子量进行了研究。实验表明，细胞内的PHB经三氯甲烷一步提取其纯度可达98％以上，而且其纯度受细胞中PHB的原始含量以及不同的前处理的影响很小，同时PHB中少量的杂质对特粘度的影响并不十分显著，因而以三氯甲烷提取液过滤后直接测定粘度是可行的。同时实验结果也表明，测定特性粘度时，浓度控制在1.25-5.00mg/ml之间，以1n(t/t_0)/C法计算特性粘度，三氯甲烷提取时温度控制在50 ℃～60 ℃，提取时间为3小时以上，得到的结果相对误差较小。同时发现PHB在30 ℃、pH > 9.0的碱性条件下是不稳定的，而在pH在2.0 ～ 4.0的偏酸和中性环境下是相对稳定的，当温度提高到65 ℃时，在pH ＝ 2.0的条件下，特性粘度随时间的延长也会逐渐降.低。还发现湿细胞中的PHB比丙酮干燥的细胞中的PHB更抗次氯酸钠，湿细胞对次氯酸钠的抗性与细胞表面的脂高PHB的纯度作用更大。但温度不宜太高，高温和过长的作用时间都会使分子量大幅度降低。从次氯酸钠、过氧化氢、triton+H_2O_2、过氧乙酸和硝酸对提取PHB作用效果比较可知，综合考虑分子量和纯度，过氧乙酸效果最好。首次发现了PHB在120 ℃－180 ℃真空加热处理后会改变其在三氯甲烷等有机溶剂中的溶解性；溶解的程度与加热处理的温度、加热时间和被处理样品的分子量有关。核磁共振、X－光晶体衍射、红外光谱及拉曼光谱对加热前后的PHB研究表明，PHB在121 ℃－180 ℃真空加热后部分或全部不溶于三氯甲烷的性质，并不是因为加热使PHB一级结构发生改变（如变联等），也不是因为结晶度的增减，而是因为加热使PHB结晶形态发生了改变。而温度对菌体内的PHB的影响因其干湿状态的不同而不同，对欧文氏菌和大肠杆菌湿菌体进行加热处理可在一定程度上提高PHB的纯度，同时也会使细胞中的PHB分子量有一定程度的降低，但都不会使菌体内PHB形成结晶；欧文氏菌和大肠杆菌干菌体内的PHB特性却有所不同，干燥后菌体内的PHB的结晶度大大增加，当在120 ℃加热后两种菌体内的PHB结晶形态都会发生改变；所不同的是大肠肝菌干菌体内的PHB加热后结晶度基本上是不变的，而欧文氏菌的PHB加热后结晶度会有所上升；从加热前后细胞内的PHB和纯PHB的X光晶体衍射图的比较可知，发生变化的衍射角的位置是相同的，说明加热前后细胞内的PHB和纯PHB结晶形态的变化也是相似的，也说明欧文氏菌和大肠杆菌干细胞内的PHB在120 ℃以上加热后用三氯甲烷提取时，提取率明显下降的原因也是由于结晶形态的改变而造成的。 |
| 英文摘要 | Using microorganism to produce biodegradable plastic is one of the most promising and effective way to deal with the "white pollution", PHB which regarded as one of the substitute of oil based plastic in the future is the polymer of β-hydroxybutyrate, its characteristics are something like polypropylene, it is biodegradable. However it suffers the key problem of high cost in production PHB on large scale and the price of the product is not competitive compared with petrochemical polymers. In order to cutting down the cost, flocculation of recombined Erwinia carotovora-Ecc13B cell from broth, influence of temperature, pH and oxidants on the separation of PHB were studied; and the method which measure the molecular weight of PHB by intrinsic viscosity was also studied; furthermore, a phenomenon was found that insolubility of PHB in chloroform can be changed by pretreated at over 120 ℃ for the first time, and the mechanism of this phenomenon was explored. Several flocculants and the conditions in which they were more effective were studied in fermentation medium of recombinant Erwinia carotovora-Ecc13B. The result showed that the concentration. In acidic condition, the flocculating effects of ZTC, J-101 and polyaluminium had the flocculating function, among these, ZTC, J-101 and Jingshuifen were the best flocculants. The relationship of spontaneous flocculation and pH of the culture fluid of transgene Erwinia carotovora-Ecc13B was studied in production of PHB. It is showed that the spontaneous flocculation occurred when pH < 4.0 or pH > 8.5. After heating at 100 ℃ for 30 minute, it lost this spontaneous flocculation character. The Zeta potential of culture fluid at different pH measured by Zeta potential analyzer. It revealed that the floculation which occurred at pH < 4.0 is related with the isoelectric precipitation of bacteria cells, and the flocculation which occurred at pH > 8.5 was related with high valence cationic exchanging into the electric double layer. The method which measure the molecular weight of PHB by intrinsic viscosity was also studied, it was discovered that the purity of PHB could reach over 98% after extracting by chloroform depositing by n-hexane for the cycle, the percentage of PHB in cell has very little influence on purity of extracted PHB, there was no significant difference among the intrinsic viscosity of different samples of PHB which was very pure or a little impure, so it was a practical way measure the intrinsic viscosity directly using the filtrate of chloroform which extracted PHB from cell, and it was revealed that, when measure the intrinsic viscosity, the relative error could be greatly reduced, while the concentration of PHB controlled at the range of 1.25 to 5.00 mg/ml; the intrinsic viscosity was calculated with the formula ln(t/t_0)/C; the temperature of chloroform was controlled at the range of 50 to 60 ℃ when extract PHB; the extracting process should be last at least 3h. The insolubility of PHB in chloroform which pretreated at over 120 ℃ and its mechanism were studied for the first time. It showed that the solubility of PHB in chloroform would be changed after it was pretreated at the range of 121 ℃-180 ℃ in 760mmHg vacuum. The degree of insolubility in chloroform of PHB was related with the temperature, time of pretreating and molecular weight of PHB. the higher the temperature was the more difficulty it solved, the longer pretreating takes the more difficulty it was solved, and the larger the molecular weight is the more difficulty it was solved. But when heated at 190 ℃ (over its melting point), it convert to soluble in chloroform again. By the characteristic of PHB which heated at 150 ℃ can solved in pyridine and and dimethyl sulfoxide, and the study of NMR, X-ray diffraction of PHB, it can be concluded that the pretreating at 150 ℃ did not result in the change of primary structure of PHB, and the insolubility of PHB in chloroform related with the change of crystal structure. The change of degree of crystallinity and crystal form of intracellular PHB which pretreated over 120 ℃ in recombinant E. coli and Erwinia carotovora cells was studied. In wet cell, the degree of crystallinity of PHB was low. Heating at 124 ℃ 1 hour could neither change the degree of crystallinity nor the crystal form of intracellular PHB. In dry cell the degree of crystallinity of PHB became high, but the rise of degree of crystallinity didn't influence its solubility in chloroform. However, when heated the dry cell at 150 ℃ for 50 minutes the crystal form of intracellular PHB changed and rate of extraction with chloroform fell down deeply. The extent of falling down of extraction rate with chloroform is related with the temperature and time of heating. It could be revealed that the PHB was relatively more stable in acid buffer than in alkali buffer. The kinetic curve of intrinsic viscosity of PHA inside E. coli cells also showed that the PHB was more susceptible to alkali than to acid, and the higher temperature the more frigate it would be at the same pH. Experimental study on the extraction of PHA from bacteria cell with inorganic solvents was carried out. Sodium hypochlorite, hydrogen peroxide, hydrogen peroxide + triton, peroxyacetic acid and nitric acid were used as extracting reagents, on account of molecular weight and purity of PHB. peroxyacetic acid was more effective. The PHB in wet cell was more stable when treated with sodium hypochlorite than that of pretreated by acetone; the resistance of PHB in wet cell to sodium hypochlorite was related to the lipid of cell surface. |
| 语种 | 中文 |
| 公开日期 | 2013-09-27 |
| 页码 | 120 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/2001]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 秦杰. 从微生物发酵液中分离提取PHB[D]. 北京. 中国科学院研究生院. 1999. |
入库方式： OAI收割
来源：过程工程研究所
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