【作者】 贾波；

【导师】 梅乐和； 金志华；

【作者基本信息】 浙江大学 ， 生物化工， 2007， 博士

【摘要】 普那霉素是由始旋链霉菌产生的一种链阳性菌素类抗生素，对包括甲氧西林耐药的金黄色葡萄球菌、万古霉素耐药的肠球菌等在内的大多数革兰氏阳性菌均具有较强的杀菌活性，且抗生素后效应较长，被专家们视为治疗顽固性革兰氏阳性菌感染的特选药物。目前，普那霉素的生产水平较低，远不能满足临床上的需求。本文将围绕如何提高普那霉素的产量和质量，对Streptomyces pristinaespiralisXC416的发酵工艺、普那霉素的分离纯化工艺等进行较为系统的研究，并探索采用树脂吸附的原位分离技术与发酵相耦合生产普那霉素的新工艺。研究了S.pristinaespiralis XC416的摇瓶发酵条件。通过单因素试验和以响应面为主的统计学方法对发酵培养基进行了优化，优化后的发酵培养基组成为：可溶性淀粉65．45g／L，葡萄糖9．30g／L，豆饼粉10g／L，蛋白胨5g／L，鱼粉10 g／L，酵母粉3 g／L，（NH₄）₂SO₄ 1．5g／L，MgSO₄·7H₂O 4．93g／L，KH₂PO₄ 0．2g／L，NaNO₃0．75g／L，CaCO₃4g／L。优化后的摇瓶培养条件为：种龄48 h，接种量8％，摇瓶装液量25 mL／250 mL，发酵初始pH值6．5～7．0，温度23℃，摇床转速为240rpm。在此条件下，S.pristinaespiralis XC416的摇瓶发酵产量达到了490．7 mg／L。以生物反应与分离耦合的集成化思路为指导，建立了以JD-1大孔吸附树脂直接从发酵液中吸附分离目标产物的原位分离技术并耦合普那霉素发酵的新方法。在S.pristinaespiralis XC416摇瓶发酵45 h时添加8％（w／v）JD-1树脂，普那霉素的产量达到了1353 mg／L，JD-1树脂对普那霉素的吸附容量约为13．5 mg／g。在5 L发酵罐上对S.pristinaespiralis XC416分批发酵的代谢规律进行了研究，确定了采用分两阶段控制溶氧水平的分批发酵工艺，使普那霉素的产量达到了604 mg／L。并在此基础上建立了普那霉素分批发酵的形态学结构模型，该模型能较好地反映普那霉素的发酵过程。同时以模糊理论为基础建立了S.pristinaespiralis XC416的模糊生长动力学模型，普那霉素发酵过程的生长生产耦合度为1．64％，为典型的非生长耦联过程。建立了对发酵液中的普那霉素进行分离纯化的硅胶柱层析方法。优化后的柱层析操作条件为：选用100～200目的硅胶，二氯甲烷：乙酸乙酯：乙醇=80：35：5（V：V）为洗脱剂，流速为2．0 mL／min，普那霉素的进样浓度为2．26g／L，硅胶的总负载量约为0．81 mg／g，温度为25℃。在此条件下分离所得产品的纯度约为96％，回收率约为97％。通过制备高效液相色谱法对硅胶柱层析分离后得到的产品进一步纯化精制，最后制备得到了纯度在99％以上的普那霉素Ⅱ_A纯品，并运用紫外光谱、红外光谱、核磁共振和质谱等多种现代波谱学方法对所制备的普那霉素Ⅱ_A纯品进行了结构鉴定。探索了采用扩张床吸附分离异位耦合发酵生产普那霉素的新工艺。采用游离菌体的耦合模式，发酵液的粘度过高，导致形成“直通槽”等不利于床层稳定的因素，阻碍了耦合操作的顺利进行。采用固定化菌体的耦合模式，虽然有利于降低发酵液的粘度，使床层较为稳定，但在启动耦合操作24 h之内发酵液即被杂菌污染，致使耦合操作仍无法顺利进行。更多还原

【Abstract】 Pristinamycins, produced by Streptomyces pristinaespiralis, is a member of streptogramin family of antibiotics. It not only has strong antibacterial activity against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus, but exhibits a prolonged post-antibiotics effect. So pristinamycins is considered as the specially selected medicament against the stubborn gram-positive bacterium infection. Up to now, the production level of pristinamycins is still so low that it could not meet the clinical requirement. In this paper, the fermentation, separation and purification techniques for pristinamycins production were all systematically studied. In addition, a new technique, the integrated fermentation and separation technology based on the in situ product removal technique by adsorbent resin, was developed to produce pristinamycins.The fermentation condition in shake flasks by Streptomyces pristinaespiralis XC416 was studied. The production medium was optimized both by ’one-variable-at-a-time’ approach and the statistical methods based on Response Surface Method. The optimized medium composition was as follows: soluble starch 65.45 g/L, glucose 9.30 g/L, soybean flour 10 g/L, peptone 5 g/L, fish flour 10 g/L, yeast extract 3 g/L, （NH₄）SO₄ 1.5 g/L, MgSO₄·7H₂O 4.93 g/L, KH₂PO₄ 0.2 g/L, NaNO₃ 0.75 g/L, CaCO₃ 4 g/L. And the optimized cultural condition in shake flasks was as follows: inoculum age of 48 h, inoculum level of 8%, medium volume of 25 mL in a 250 mL shake flask, initial pH value of 6.5-7.0, temperature of 23℃, shaking speed of 240 rpm. Under the above conditions, S. pristinaespiralis XC416 could produce 490.7 mg/L pristinamycins in the shake flask.Taking the integrated bioreaction and separation process as guide, a new technique, viz. the technique of in situ product removal based on the adsorption of the product by macroreticular resin JD-1 directly from the fermentation broth, was developed for pristinamycins production. When 8% （w/v） resin JD-1 was added into the broth at 45 h during the fermentation process, the yield of pristinamycins could reach 1353 mg/L and the adsorption capacity of resin JD-1 on pristinamycins was about 13.5 mg/g.The batch fermentation of Streptomyces pristinaespiralis XC416 in 5 L bioreactor was studied, and the yield of pristinamycins reached 604 mg/L by controlling the different dissolved oxygen levels during the fermentation process. On the other hand, a morphologically structured model was developed, well simulating the batch fermentation process for pristinamycins production. Also, a fuzzy growth model of Streptomyces pristinaespiralis XC416 was developed based on the fuzzy theory. From the model, the coupled degree of the cell growth and the pristinamycins production was 1.64%, indicating that it is a non growth-associated process.The separation and purification of pristinamycins from the fermentation broth by the method of silica gel column chromatography was developed. And the operational condition was as follows: 100-200 mesh silica gel to be the stationary phase, solvent system of methlene chloride: ethyl acetate: ethanol = 80:35:5（V:V） to be the eluent, the flow rate of the eluent to be 2.0 mL/min, the feeding concentration of pristinamycins to be 2.26 g/L, the total loading amount of the silica gel to be 0.81 mg/g and the temperature to be 25℃. Under the above condition, the product with the purity of about 96% and the recovery of about 97% was finally obtained. After then, by the preparative HPLC method to purify and refine the product obtained above further, the final product, viz. pristinamycinⅡ_A with the purity of more than 99% was obtained. And the chemical structure of pristinamycinⅡ_A was elucidated by UV, IR, NMR and MS methods.The exploring of a new technique to produce pristinamycins by using the integrated bioreaction and separation process based on expanded bed adsorption technique was conducted. When using the integrated mode of free cell, the high viscosity of the broth could easily cause some negative factors, such as the liquid channels, hampering the successful operation. And when using the integrated mode of immobilized cell, although the viscosity of the broth was effectively reduced and the expanded bed was stabilized, the broth was contaminated by some bacteria in 24 h after the onset of the integrated operation, also hampering the successful operation.更多还原