卡那霉素A、B组分分离纯化新技术研究

【作者】 李忠琴；

【导师】 郭养浩；

【作者基本信息】 福州大学 ， 生物化学与分子生物学， 2002， 硕士

【摘要】 目前国内卡那霉素生产过程大多采用732~#强酸性阳离子交换树脂静态吸附、动态洗脱工艺。针对该工艺存在的缺陷，本工作进行卡那霉素A、B组分分离纯化新技术研究。 卡那霉素A、B属于多元弱碱水溶性化合物。本工作分别采用基于缓冲体系原理的分段线性回归分析、联立方程的算法和基于酸碱滴定通式的Rosenbrock算法，处理卡那霉素A、B电位滴定数据。估算得到的卡那霉素A多元离解常数与文献报道的数值吻合。本论文首次全面报道了卡那霉素B的离解常数：根据卡那霉素A、B的电离度模拟不同pH条件下各价态的分布系数曲线(pH-分布系数曲线)。 以发酵液为原料，筛选了八种不同类型强酸性和弱酸性阳离子交换树脂。弱酸性大孔型D-186树脂具有卡那霉素吸附总量高、杂质吸附量少和卡B组分富集率高的特点。运用正交试验设计法优化了上柱吸附富集条件(pH7.0，流速2.0V／V／h)和杂质洗脱条件(硫酸铵溶液浓度1.0％(v／v)，pH7.5，流速1.25V／V／h)。采用D-186树脂和732树脂四柱串联动态吸附洗脱工艺流程，可以有效富集发酵液中的卡那霉素B组分。在发酵不正常时，能够保证卡碱产品的质量。 采用D-186树脂从结晶母液中分离卡那霉素A、B组分。运用正交试验设计法优化上柱吸附富集条件(母液稀释浓度1.26万u／ml,pH7.0,流速1.5VN／h)和杂质洗脱条件(硫酸铵溶液浓度1.5％(V／V)，pH7.5，流速1.75V／V／h)。采用三柱串联动态吸附洗脱工艺路线，使卡那霉素A、B组分的总回收率分别达到80％和93％以上，杂质去除串达到99％。 将从发酵液和结晶母液中富集的卡那霉素B组分枓液浓缩后按比例混合(浓度30～35万u／ml，卡B组分含量约占35％)，采用阴离子交换树脂H401进行柱层析分离。优化了层析分离操作条件：①粒径80目(0.20mm)，交联度2.5的H401阴离子交换树脂：②上柱量为1.05ug/ml：③洗脱剂NH4OH浓度0.6％(V／V)：④洗脱流速0.5ml／min。卡A产品纯度达到99.5％，卡B产品纯度达到90％以上。更多还原

【Abstract】 The domestic current Kanamycin A separating technology is static adsorption and dynamic eluation processing based on 732 strong acid cation-exchange resin. A new extraction and purification technology of Kanamycin A and B was developed in this work.Kanamycin A and B are soluble weak polybases. According to the determined data in our potentiometric titration experiments ,the polybasic ionization constants of Kanamycin A and B were estimated by means of two algorithm, Stepwise Linear Regression and Simultaneous Equations Algorithm and Rosenbrock Algorithm. The calculated polybasic ionization constants of Kanamycin A were similar to the reported data. The stepwise ionization constants of Kanamycin B pKa,=5.61, pKa2=6.56, pKa3=7.43, pKa4=8.22, pKaj =9.11, were first reported. The distribution of different Kanamycin A and B charged-ion in different pH conditions was simulated.After comparison test of eight cation-exchange resins , D-186 cation resin was sorted out to separate Kanamycin B from the fermented broth, which was characterized of higher adsorption rate of Kanamycin B compared with Kanamycin A and lower impurity adsorption rate. The orthogonal design and analysis of experiments was applied to optimize the operation condition during adsorption ( pHV.O, flow rate 1.5V/V/h. ) and the condition during impurity elution ((NH4)2SO concentration 1.0%(VAO, pH7.5, flow rate 1.25V/V/h ). A new technology of dynamic absorption and dynamic eluation involving one D-186 resin and three 732 resins column was developed, which was able to concentrate Kanamycin B effectively and guarantee the quality of final product of Kanamycin A in abnormal fermentation.D-186 resin was also selected to separate Kanamycin A and B from crystallization residue. Then the optimum operation condition of extracting Kanamycin and purging away impurity is also determined. The concentration of diluted crystallization residue was 12600 u/ml, pH 7.0, flow rate 1.5V/V/h; and the concentration of (NH4)2SO4 was 1.5%(V/V), pH 7.5, flow rate 1.75V/V/h. The dynamic absorption and dynamic eluation technology incuding three columns of D-186 resin was established. The recovery rate ofKanamycin A and B was respectively 80% and 93%. The elimination rate of impurity was about 99%.The collected fractions Kanamycin B from fermented broth and crystallization residue solution weremixed and then concentrated to 30-35 X 105u/ml, contained about 35% Kanamycin B. Based on the principle of separation chromatography, Kanamycin B was separated and purified by H401 anion-exchange resin. The optimum technological conditions were determined: the diameter of resin 0.20mm, the linked-degree of resin 2.5 , and the adder quantities 1.05ug/ml (resin), the eluant concentration of NH4OH 0.6%(V/V), the elution rate 0.5 ml/min for a column of 1> 1.8cm X 300cm. The purity of Kanamycin A\B was 99.5% and 90%.更多还原