运用全细胞催化法生产α-酮戊二酸的过程中,α-酮戊二酸生物转化液中含有残留底物、杂蛋白、有机杂酸和色素等杂质,用单一的分离方法不易去除。该文使用钙盐沉淀法结合离子交换法对α-酮戊二酸全细胞转化液进行分离纯化,将预处理后的料液酸化至pH=2.0,用Ca(OH)2做沉淀剂沉淀α-酮戊二酸得到α-酮戊二酸钙,然后使用浓H2SO4处理α-酮戊二酸钙得到离子交换母液,经液相色谱检测,α-酮戊二酸的纯度从78.79%上升至93.66%。通过离子交换静态试验筛选出D301型树脂作为离子交换填料。动态上样最优参数为:高径比2.5∶1、上样质量浓度30 g/L、上样流速2.4 BV/h。最佳洗脱条件为:质量分数为0.02%的HCl溶液洗脱2个柱体积,流速4 BV/h,以质量分数为5%的NaOH溶液洗脱4个柱体积,流速2 BV/h,最终分离纯化处理后的总收率为80.32%,纯度为97.32%。
During the process of producing α-ketoglutaric acid by whole cell transformation, the biotransformation solution contains impurities, such as residual substrates, macromolecular proteins, organic heteroacids and pigments, which is difficult to remove by a single separation method. In order to purify α-ketoglutaric acid from whole cell transformation solution, the calcium salt method and ion exchange method were together used for separation and extraction process of α-ketoglutaric acid. The pretreated whole cell transformation stock solution was acidified to pH 2.0, and calcium α-ketoglutarate was formed by precipitation of α-ketoglutaric acid with Ca(OH)2 as the precipitant. The ion-exchange master batch was obtained by treatment of calcium α-ketoglutarate using concentrated H2SO4. The purity of α-ketoglutaric acid increased from 78.79% to 93.66%, analyzed by HPLC. The D301 anion resin was selected as ion exchange filler by ion exchange static test. The optimal parameters for dynamic loading showed as follows: the height to diameter ratio of 2.5∶1, loading concentration of 30 g/L and loading flow rate of 2.4 BV/h. The optimal elution conditions were: 2 BVs eluted by 0.02% mass fraction of HCl at a flow rate of 4 BV/h, 4 BVs eluted by 5% mass fraction of NaOH at a flow rate of 2 BV/h. The total yield of α-ketoglutaric acid after separation and purification process was 80.32% and the chromatographic purity reached 97.32%.
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