【作者】 徐婕；

【导师】 朱家文；

【作者基本信息】 华东理工大学 ， 化学工程， 2012， 博士

【摘要】 异戊酰螺旋霉素是由将克隆自碳霉素产生菌的4"-O-异戊酰基转移酶基因整合到螺旋霉素产生菌Streptomyces spiramyceticus F-21的染色体上而构建成的一株稳定的生物工程菌WSJ-1-195产生的具有一系列相似结构的混合物,由于其活性高、稳定性好以及相对耐药性好等特点,而成为主要的抗生素产品之一,其中,异戊酰螺旋霉素Ⅲ具有最好的稳定性和最高的活性。但由于异戊酰螺旋霉素发酵液中存在与异戊酰螺旋霉素Ⅲ(主要抗菌活性成分)相似的杂质组分,采用常规的分离技术难以获得高纯度的异戊酰螺旋霉素Ⅲ产品,成为制约异戊酰螺旋霉素Ⅲ工业化生产的瓶颈之一本文针对异戊酰螺旋霉素Ⅱ和异戊酰螺旋霉素Ⅲ组分的分离难点,以提高异戊酰螺旋霉素Ⅲ组分的纯度和收率为目标,对分离纯化过程进行了系统的研究以及模拟计算,提出了间歇柱层析分离工艺路线,以解决异戊酰螺旋霉素Ⅱ和异戊酰螺旋霉素Ⅲ组分分离的关键技术难题。本文的主要内容和结论如下：(1)以吸附剂对异戊酰螺旋霉素Ⅲ组分的选择性系数为主要指标,分别考察了吸附剂、离子强度及pH对异戊酰螺旋霉素Ⅲ选择性吸附效果的影响。比较筛选得出较优吸附剂和吸附条件：以异戊酰螺旋霉素粗品为原料,HZ820树脂为吸附剂,离子强度0.5 mol/L、pH 6.5时,对异戊酰螺旋霉素Ⅲ组分的选择性系数较高(KⅡⅢ为1.67),表现出较好的吸附选择性。根据热力学计算可知,该过程为自发熵增的吸热过程。(2)测定了不同温度、pH及离子强度下,异戊酰螺旋霉素Ⅱ、Ⅲ组分在HZ820大孔吸附树脂上的竞争吸附平衡数据。实验范围内,温度、pH及离子强度的增加均对异戊酰螺旋霉素Ⅱ、Ⅲ组分的吸附过程有利。分别采用了扩展Langmuir模型、扩展Langmuir-Freundlich模型和扩展Jovanovic-Freundlich模型对平衡数据进行拟合和对比,其中扩展Langmuir-Freundlich模型取得了最好的拟合结果。根据扩展Langmuir-Freundlich吸附等温线模型的拟合结果,对该模型进行了误差分析,其相对误差在15%以内,说明该模型能够较好的描述异戊酰螺旋霉素Ⅱ、Ⅲ组分在HZ820树脂上的竞争吸附行为。(3)测定了异戊酰螺旋霉素在HZ820树脂上的间歇吸附动力学数据,考察了温度、pH及离子强度等因素对动力学过程的影响,分别采用拟一级、拟二级和颗粒内扩散模型对比拟合异戊酰螺旋霉素在HZ820大孔吸附树脂上的吸附过程,结果表明该过程为一个非均相的优惠吸附过程,吸附速率由液膜扩散和颗粒内扩散综合控制。(4)研究了进料流速、浓度、床层高径比等因素对固定床内异戊酰螺旋霉素在HZ820大孔吸附树脂上的吸附性能的影响,采用同时考虑液膜扩散阻力、孔内扩散阻力和轴向扩散阻力的综合速率模型对吸附过程进行模拟,计算得到相关动力学参数,并对模型进行了误差分析。结果表明：进料流速和浓度的增加对异戊酰螺旋霉素固定床吸附过程不利,床层高径比的增加可以提高固定床吸附效率；液膜扩散阻力和孔内扩散阻力均对异戊酰螺旋霉素固定床吸附过程有重要影响,轴向扩散影响较小；模型的相对误差在10%以内,说明液膜、颗粒内扩散及轴向扩散综合速率模型较好的描述了异戊酰螺旋霉素在HZ820树脂上的固定床吸附行为。(5)在前述研究的基础上,以异戊酰螺旋霉素粗品为原料,研究了固定床吸附和解吸过程。从采用前述优选的HZ820吸附剂和吸附条件时测得的异戊酰螺旋霉素Ⅱ和异戊酰螺旋霉素Ⅲ组分的穿透曲线,吸附过程中异戊酰螺旋霉素Ⅱ和异戊酰螺旋霉素Ⅲ组分表现出了明显的竞争吸附特性；随后采用石油醚为洗脱剂,以1.20 BV/hr流速洗脱,洗脱剂用量6.00 BV时,对异戊酰螺旋霉素Ⅲ的洗脱选择性(KⅡⅢ为1.60)和收率(94.2%)均较好。(6)以异戊酰螺旋霉素发酵液为原料,经过膜过滤、树脂吸附、树脂洗涤、树脂洗脱、结晶等步骤制备异戊酰螺旋霉素粗品。研究和优化了各单元操作的工艺条件和实际效果,采用pH 8.0,1=0.2 mol-L-1的磷酸盐缓冲液对吸附完毕的树脂进行杂质洗涤,选用石油醚为洗脱剂,对洗涤完毕的树脂进行洗脱,可使异戊酰螺旋霉素Ⅲ得到富集的同时,对异戊酰螺旋霉素Ⅱ组分有一定的去除作用,从而得到高品质的异戊酰螺旋霉素产品。本文针对国内菌种的异戊酰螺旋霉素发酵液中存在副产物组分多,浓度高,和主要产物异戊酰螺旋霉素Ⅲ之间分离的难点,提出了一条分离纯化异戊酰螺旋霉素Ⅲ的新工艺,研究成果为异戊酰螺旋霉素Ⅲ生产工艺的开发和设计提供了理论和实验基础。该技术与膜过滤、结晶等技术组合可形成先进的异戊酰螺旋霉素提取分离生产工艺,能够显著提高异戊酰螺旋霉素Ⅲ产品的质量和收率,具有广阔的发展前景。更多还原

【Abstract】 4"-O-isovalerylspiramycin is a new macrolide antibiotic produced by genetically engineered Streptomyces spiramyceticus transformed with the 4"-O-acyltransferase gene from S. mycarofaciens.4"-O-isovalerylspiramycin is a mixture of 4"-O-isovalerylspiramycin I, II, and III, which all possess similar molecular structures. During fermentation, 4"-O-isovalerylspiramycinⅢis the main and active product. However, separation of 4"-O-isovalerylspiramycinⅡandⅢis very difficult because they have very similar chemical structures and characteristics.In the present work, the separation process of 4"-O-isovalerylspiramycinⅡand III has been studied systematically. According to the difficulties of separation and purification, an innovative chromatographic technology routine has been presented, which can solve the difficulties in the separation of 4"-O-isovalerylspiramycin II and III.The main contents and conclusions are listed as follows:(1)With impure 4"-O-isovalerylspiramycin (4"-O-isovalerylspiramycinⅡ:Ⅲ=2:3, w/w) as raw material, the appropriate selective adsorption conditions for 4"-O-isovalerylspiramycinⅢ, which have been obtained by batch adsorption experiments, are as follows:HZ820 as adsorbent, ionic strength 0.5 mol/L, pH 6.5, KⅡⅢis 1.67, indicating that HZ820 has a good selectivity for 4"-O-isovalerylspiramycin III under these conditions. According to thermodynamics results, this adsorption is a spontaneous, entropy increase and endothermic process.(2)The competitive adsorption equilibrium data for the binary system of 4"-O-isovalerylspiramycinⅡandⅢonto a macroporous resin HZ820 were tested and modeled using the extended Langmuir, extended Langmuir-Freundlich and extended Jovanovic-Freundlich equation at different pH, ionic strengths and temperatures. For both 4"-O-isovalerylspiramycinⅡandⅢ, the adsorption was enhanced with the increase of pH, ionic strength and temperature. The extended Langmuir-Freundlich adsorption model gave the best competitive isotherm fits for both 4"-O-isovalerylspiramycin II and III. The relative errors of the extended Langmuir-Freundlich model were within 15%.(3)The effects of the operation parameters, which is mentioned above, on the adsorption kinetics of 4"-O-isovalerylspiramycin to HZ820 resin have been determined. Pseudo-first-order, Pseudo-second-order and intra-particle diffusion model each have been used to describe 4"-O-isovalerylspiramycin adsorption profile in thermostatic shaker. The results showed that Pseudo-second-order model successfully described the adsorption of 4"-O-isovalerylspiramycin on HZ820. The adsorption was favorable process.(4)The effect of flow velocity, feed concentration, and aspect ratio on the fixed-bed adsorption characteristics of 4"-O-isovalerylspiramycin was investigated by using a macroporous resin HZ820 as adsorbent. The film, porous and axial diffusion model taking the film mass transfer, pore diffusion and axial dispersion into account was adopted to describe the breakthrough curves. The mass transfer coefficients and the relative errors of the model were consequently determined. The results showed that the increase of flow velocity and feed concentration was not in favor of the fixed-bed adsorption performance, while the increase of aspect ratio could improve the mass transfer. It is clear that both liquid film mass transfer and pore diffusion play important roles in the fixed-bed adsorption of 4"-O-isovalerylspiramycin onto HZ820, whereas the axial dispersion could be negligible in the experimental range. The relative errors of the model were within 10%, indicating a satisfactory simulation.(5)On the basis of those above, the adsorption and desorption processes with impure 4"-O-isovalerylspiramycin as raw material have been studied by using the optimal conditions in fixed bed. It shows that a competitive adsorption between 4"-O-isovalerylspiramycin II andⅢoccurred; petroleum ether as eluent, flow velocity 1.20 BV/hr, the elution volume 6.00BV, KⅡⅢis 1.60, and thus 4"-O-isovalerylspiramycin III desorption rate can reach 94.2%.(6)A complete set of separation process for recovering 4"-O-isovalerylspiramycin III from fermentation broth was introduced, including membrane filtration, adsorption, impurity washing, desorption and crystallization in the eluate. The technological conditions and its practical effect of each unit operation were studied and optimized. The phosphate hydrate buffer solution (pH 8.0,1=0.2 mol·L-1)was chosen to wash impurities. Through the whole separation process, the content of 4"-O-isovalerylspiramycin III in product could meet the requirement.Aim at the difficulties of separation of 4"-O-isovalerylspiramycin II and III, an innovative technology routine for separation and purification of 4"-O-isovalerylspiramycin III has been presented in this paper. This research provides the experimental foundation for the development and design of large-scale industrial equipment. With the combination of membrane filtration and crystallization, an advanced 4"-O-isovalerylspiramycin production technology can be established, which can improve the purity and yield of 4"-O-isovalerylspiramycin III significantly and be of great prospect in practical application.更多还原