S-腺苷-L-甲硫氨酸的分离纯化及其稳定盐的制备过程

【作者】 赵晓星；

【导师】 岑沛霖；

【作者基本信息】 浙江大学 ， 生物化工， 2006， 硕士

【摘要】 S—腺苷—L—甲硫氨酸（S-adenosyl-L-methionine,简称SAM,SAMe或AdoMet）是一种重要的中间代谢产物,广泛存在于各种生物体中,也是人体内一种重要的生理活性物质。本文主要研究了温度及pH对SAM稳定性的影响;SAM的分离纯化;SAM硫酸盐,SAM对甲苯磺酸硫酸双盐,SAM1,4—丁二磺酸盐的制备;SAM硫酸盐,SAM对甲苯磺酸硫酸双盐的热稳定性。 对SAM稳定性进行研究表明,在4℃的条件下,pH从3.0上升到7.0,SAM的降解速率常数k_d由1.810×10^-3 gL^-1h^-1增加到4.817×10^-3 gL^-1h^-1,降解速度随pH升高而加快;在pH=3.0的条件下,温度从50℃增加到70℃,降解速率常数kd从4.44×10—2 gL-1h-1增大到7.11×10—1 gL-1h-1,随温度提升迅速加快。 采用离子交换法从细胞提取液中分离纯化SAM。此法相对于传统的使用苦味酸,苦酮酸选择沉淀SAM的方法,提高了SAM的回收率,而且减小了操作的危险性和对环境的污染。研究得出SAM提取液在JK110弱酸性离子交换树脂上的最佳上柱条件为:SAM浓度:4.98g/L;流速:2BV/h;SAM料液pH:5.0。以硫酸作为洗脱剂洗脱SAM,得出最佳洗脱条件为0.1mol/L的硫酸,流速2BV/h。流速对洗脱效果基本没有影响。SAM洗脱效率为91.9%。 采用有机反相沉淀的方法制备SAM硫酸盐,并在此基础上优化SAM对甲苯磺酸硫酸双盐的制备工艺,解决了以往稳定盐制备中结晶收率低,纯度低的问题。所得SAM硫酸盐产品收率为97.0%,纯度92%;SAM对甲苯磺酸硫酸双盐收率为87.5%。对两种稳定盐的热稳定性进行研究表明,SAM对甲苯磺酸硫酸双盐的热稳定性得到极大的改善。在45℃的条件下,30天后,SAM硫酸盐中有效成分降解了61.0%,而在SAM对甲苯磺酸硫酸双盐中,仅降解了18.4%。 在1,4—丁二磺酸二钠盐的制备过程中由于表面活性剂EDTA的加入,反应时间由文献中的72h缩短到3h,反应转化率达到99.39%。此外,采用HPCE建立1,4—丁二磺酸的检测方法。该方法的线性相关系数0.9989,日内变异系数0.05%,日间变异系数0.9%,回收率96.30%。该方法准确性高、重现性好、灵敏度高,非常适合于1,4—丁二磺酸二钠盐含量的测定。以1,4—丁二磺酸洗脱SAM。得出浓度为0.2mol/L为合适的洗脱浓度,洗脱峰集中,洗脱效率为96.17%;流速对洗脱效率影响不大。收集洗脱液,过大孔吸附树脂柱后经喷雾干燥得到SAM1,4—丁二磺酸产品。 本文旨在开发并完善已有的SAM分离纯化工艺,建立一条低成本,高产率的SAM稳定性盐的制备途径,为SAM工业化应用打下良好基础。更多还原

【Abstract】 S-adenosyl-L -methionine （SAM, also known as Ado-Met, SAMe）, an important product of intermediate metabolism, is widely found in various organisms. The influence of temperature and pH on SAM, the purification and preparation process of SAM with its stable salts SAM sulphate, SAM·p-toluenesulfonate sulphate and SAM·1, 4 -butanedisulphonate salt, the thermal stability of SAM sulphate and SAM ·p-toluenes -ulfonate sulphate were studied in this paper.The study on the stability of SAM indicated, under the condition of 4℃, the experiment had shown that from pH 3.0 to pH 7.0, k_d increased correspondingly from 1.810×10^-3 gL^-1h^-1 to 4.817×10^-3 gL^-1h^-1; under the condition of pH3.0, the degradation velocity constant k_d increased from 4.438×10^-2 gL^-1h^-1 to 7.11×10^-1 gL^-1h^-1 when the temperature increased from 50℃ to 70℃. It was obviously that the SAM degradation velocity was affected both by the temperature and the pH. So during the process of purification of SAM, it was necessary to keep operation under low temperature and pH to avoid the degradation of SAM.Compared with the traditional way of using picric acid or picrolonic: acid to precipitate SAM selectively, this method not only increased the recovery, but also diminished the operation risk and the environmental pollution. It was shown in the study that the optimal absorption condition of SAM in the JK110 weak cation exchange resin was: concentration of SAM solution: 4.98g/L; flow rate: 2BV/L; pH5.0. Using sulphuric acid to elute SAM and the suitable elution condition was found as follows: sulphuric acid concentration: 0.1mol/L, flow rate: 2BV/h. Flow rate had little influence on elution effectivity. The recovery of SAM reached 91.9%.The reversal phase precipitation method was involved in the preparation of SAM sulphate and SAM·p-toluenesulfonate sulphate which improved the crystallization yield and product purity. The yield of SAM sulphate reached 97.0% with the purity of 92%; the yield of SAM· p-toluenesulfonate sulphate reached 87.5%.It was found that the thermal stability of SAM· p-toluenesulfonate sulphate was greatly improved. Under the condition of 45℃, the active composition in SAM sulphate reduced by 61.0% while 18.4 in SAM· p-toluenesulfonate sulphate after 30 days.In the addition of surfactant EDTA of the reaction system the reaction time was shortened from 36 hours in the literature to about only 3 hours. The product yield was 99.39%. Besides, an analytic method based on the high performance capillaries electrophoresis （HPCE） was established. The precision, recovery and repeatability ofthe methods were evaluated. The results showed that the linear correlation coefficient for the method was 0.999, and recovery ratio was 96.5%.Coefficients of variation within and between days were 0.8% and 0.5%, respectively. The established analytic method was fast, accurate and repeatable, which had been successfully applied in the synthesis process of 1, 4-butanedisulphonic disodium salt.Using 1, 4-butanedisulphonic acid to elute SAM and get the optimal elution condition as follows: the concentration of 1, 4-butanedisulphonic acid: 0.2mol/L. By this condition the eluting peak centralized and the elution efficiency reached 96.17%. Flow rate had little influence on the efficiency. The previous obtained eluate passed through a column containing macroporous absorption resin to remove the excessive 1, 4- butanedisulphonic acid. The collected eluate went under spray drying to get the product.This paper aimed at developing and improving the SAM purification method, establishing an effective way to prepare SAM stable salts with lower cost and higher yield.更多还原