【作者】 齐祥明；

【导师】 姚善泾；

【作者基本信息】 浙江大学 ， 生物化工， 2004， 博士

【摘要】 以高附加值蛋白质的分离、纯化为背景，在新近兴起的加压CO₂-水溶液体系等电沉淀技术的基础上，引入助沉淀剂——乙醇，形成加压CO₂-乙醇-水溶液体系沉淀蛋白质技术。该方法具有环境友好、过程可控性强等特点，符合生物分离技术的发展趋势，具有潜在的实用价值。 首先，以碱性蛋白酶2709^#粗品为对象初步考察了加压CO₂-乙醇-水溶液体系沉淀蛋白质技术的可行性和规律。结果表明，用该技术可以实现对碱性蛋白酶2709^#粗品的除杂。体系pH值较低时，会出现酶分解的现象，但通过控制操作条件可保持酶的相对稳定。CO₂压力、酶液的初始pH值、酶浓度和乙醇含量会影响溶液pH的变化和沉淀现象；操作温度对其影响不大。处理时间、CO₂压力、酶液的初始pH值、酶浓度、乙醇含量等参数会影响操作过程中蛋白质的稳定性。 第二，通过考察温度、初始pH值、乙醇含量三个参数对加压CO₂-乙醇-水三元体系的影响得知，温度、乙醇浓度对体系pH值随压力变化情况影响不大。用简化的液相pH计算模型对加压CO₂-乙醇-水三元体系液相pH进行分析，结果表明，乙醇降低了液相中CO₂和水的活度，揭示了乙醇促进CO₂的溶解但对体系pH值随压力变化情况影响不大的原因。不同浓度的乙醇水溶液在加压CO₂条件下的体积膨胀结果表明，当乙醇含量不超过30％时，体积膨胀现象及其微弱。这表明蛋白质在这样的体系中沉淀时，加压CO₂的抗溶剂效应很小。 第三，通过对BSA、溶菌酶在加压CO₂-乙醇-水体系中的沉淀情况和液相pH变化情况考察发现：BSA会在等电点附近沉淀出来，并在溶液pH进一步降低时返溶；实验考察范围达不到溶菌酶的等电点11.0，溶菌酶不沉淀，而溶菌酶粗品中等电点在5左右的杂蛋白可以沉淀出来。这表明体系中的沉淀作用主要是等电沉淀，在实验考察范围内加压CO₂抗溶剂沉淀作用不明显。 第四，乙醇具有助沉淀的作用。蛋白质分子结构数据表明，乙醇还在一定程度上改变了蛋白质分子的表面结构，这影响到了蛋白质的缓冲能力等特性，从而乙醇对加压CO₂-乙醇-水-蛋白质多元体系的pH变化有降低的作用，这使体系可处理的蛋白质浓度较加压CO₂-水-蛋白质体系大幅度提高。而在实验考察的乙醇浓度范围内，这种结构的变化是可逆的，对生物质活性的影响不大。对于加压CO₂-乙醇-水-蛋白质多元体系，入釜前pH的调节能起到改变pH的可调范围和浙江大学博士学位论文提高pH控制精度的作用。 第五，蛋白质所具有的缓冲能力使含蛋白质的多元溶液体系较不含蛋白质的三元溶液体系在相同的COZ压力下pH值有所提高，通过计算发现对于不同的CO:压力，这种pH值的增高比率近似为一常数。乙醇的加入，降低了蛋白质的缓冲能力，这一常数也有所下降。将这一规律与简化的pH计算模型结合起来，可使加压COZ一乙醇一水一蛋白质多元体系中的PH值预测大大简化，因而对于含蛋白质溶液的 pH计算和该技术在工程上的应用将非常有意义。 第六，综合四种酶类（碱性蛋白酶、溶菌酶、纤维素酶、好激酶）的研究结果发现，不同酶类在体系中稳定性有所差异，但各操作条件对稳定性的影响作用是相似的。温度、乙醇含量的增高对蛋白质稳定性有不利的影响;而增加溶液中酶浓度有增加酶的稳定性的作用。一般而言，在适当的操作范围内都能使酶保持相对稳定（相对酶活保持在80%以上）而同时得到较好的沉淀效果。对纤维素酶和叫激酶粗品进行了初步分离实验，在实验考察范围内的最佳操作条件下得到了令人满意的结果。对于纤维素粗酶，可将绝大部分CMC酶沉淀出来;而对于妇激酶可将其他杂蛋白沉淀出来。 总的来说，加压COZ一乙醇一水溶液沉淀蛋白质技术具有诸多优点和良好的发展前景，而本文从理论和应用两方面对这一过程进行了有益探讨，希望能对该过程的发展起到推动作用。更多还原

【Abstract】 On the background of the separation and purification of high-value protein products, precipitation assistant, ethanol, was introduced into the isoelectric precipitation process using compressed CO2 as a volatile acid, which was put forward recently. A precipitation process of proteins from ethanol-water solution using compressed CO2 as a volatile acid was presented, which was characterized as environment friendly and readily controllable. In a word, the process is in accord with the development of bioseparation, and has the potential to be applied in the downstream processing of proteins.Firstly, feasibility and rules of the protein precipitation process were preliminarilyinvestigated where alkaline proteinase 2709 were used as a model protein. The results indicated that the process could be used to remove impurity from the crude alkaline proteinase. The enzyme would decompose while pH value of the solution was lower. However, the enzyme remained stable at certain operating conditions. Pressure of CO2, initial pH of solution, enzyme concentration, and ethanol content have influences on the precipitation and the change of pH, while temperature has little effect. Processing time, Pressure of CO2, initial pH of solution, enzyme concentration, and ethanol content influences the stability of protein in the process.Secondly, the effects of temperature, initial pH, and ethanol content on the ternary system of compressed CO2-ethanol-water were determinated. It showed that the curves of pH as a function of pressure was little influenced by temperature and ethanol content. The pH-pressure curve of the CO2-ethanol-water ternary system was analyzed with the simplified pH calculation model, and the result indicated that ethanol decreased the activities of CO2 and water, which revealed the reasons why ethanol changed the pH-pressure curve little although it increased CO2 solubility in solution. The expansion results of ethanol-water solutions under compressed CO2 demonstrated that liquid phase expanded tinily when ethanol content was less than 30% (wt). It was concluded that the antisolvent effect of CO2 contributes little to proteins precipitation in such a system.Thirdly, the precipitation phenomena and pH-curves of the compressed COi-ethanol-water system with BSA or lysozyme were studied, and it was found that: BSA would precipitate when pH of solution decreased close to isoelectric point, and the precipitate would redissolve when pH decreased further. As to lysozyme, the pi, 11.0, couldn’t be reached within the experimental range, so lysozyme couldn’t be precipitated, but the precipitation of impurities in crude lysozyme with pi of about 5.0 could be obtained. All the results implicated that the precipitation mainly attributed to isoelectric precipitation, the action of antisolvent precipitation could not be a dominant factor in the experiments investigated.Fourthly, ethanol acts as a precipitating aid in the process. The data of proteins conformation implied that ethanol changed the conformation to some extent also. As a result, characteristic of proteins such as buffer capacity altered. Since ethanol could drive down the pH curve of the compressed CCh-ethanol-water-protein multi-component system, lower pH could be reached with the same protein concentration in this system than in compressed CCV water-protein system correspondingly, and higher protein concentration could be dealt with. However, in the experiments investigated the changes of conformation are reversible, such contents of ethanol have little negative effect on the enzymes activities. In the compressed COi-ethanol-water-protein multi-component system, the regulation of initial pH changed the adjustable pH extent, and improved pH controllability.Fifthly, the buffer capacity of proteins made pH of solution with proteins higher than that of solution without protein under the same pressure. It was found that the pH ratio of solution with protein to solution without protein remained constant with increasing of CC2 pressure. Ethanol reduced buffer capacit更多还原