Production and separation of formate dehydrogenase from Candida boidinii
Production and separation of formate dehydrogenase from Candida boidinii
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DOI：
10.1016/j.enzmictec.2006.07.035
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发表时间：
2007-03-05
影响因子：
3.4
通讯作者：
Yang, Shang-Tian
中科院分区：
文献类型：
--
作者：
Bai, Yunling;Yang, Shang-Tian
以甲醇为诱导剂，在40-1型生物反应器中生产FDH，最终胞内FDH活性达到35 U/g。在不同的渗透方法中，相对少量的甲苯处理导致细胞FDH活性最高。超声波破壁得到的粗FDH细胞提取物用聚乙烯亚胺（PEI）处理，使FDH与其他蛋白质分离。在细胞提取物中加入低浓度0.04 mg/ml的PEI，类似50%的蛋白质与PEI形成聚集体并沉淀；然而，外佣没有在这些聚集体中，而是留在溶液中。经PEI处理后，特异FDH活性提高1.6倍。SDS-PAGE分析表明，PEI沉淀去除了一些以Sepharose-Procion Blue HERB为分离配体亲和层析无法分离的杂质蛋白分子，从而提高了分离效率。亲和柱上吸附的FDH用KCl溶液洗脱。在0.2 M KCl溶液中添加5 mM NAD（+）改善了FDH的洗脱，比1 M KCl溶液的FDH比活性提高了1.38倍。总体而言，PEI沉淀和染料亲和色谱工艺获得了56%的高回收率，细胞粗提取物的比FDH活性提高了5.5倍。(c) 2006爱思唯尔公司版权所有。
The production of FDH in a 40-1 bioreactor was carried out with methanol as the inducer to reach the final intracellular FDH activity of 35 U/g cell. Among different permeabilization methods studied, treatment with toluene at a relatively small amount resulted in the cells with the highest FDH activity. Crude FDH cell extract obtained by ultrasonically breaking down the cell wall was treated with polyethyleneimine (PEI) to separate FDH from other proteins. By adding PEI at a low concentration of 0.04 mg/ml to the cell extract, similar to 50% of the proteins formed aggregates with PEI and precipitated; however, FDH was not in these aggregates and remained in the solution. After the PEI treatment, the specific FDH activity increased by 1.6-fold. SDS-PAGE analysis showed that PEI precipitation removed some impurity protein molecules that cannot be separated by affinity chromatography with Sepharose-Procion Blue HERB as the separation ligand, and thus improved the separation efficiency. The adsorbed FDH in the affinity column was eluted with KCl solution. Adding 5 mM NAD(+) in 0.2 M KCl improved the FDH elution and increased the specific FDH activity by 1.38-fold as compared to elution with 1 M KCl. Overall, the PEI precipitation and dye affinity chromatographic process obtained a high recovery yield of 56% with a 5.5-fold increase in the specific FDH activity from the crude cell extract. (c) 2006 Elsevier Inc. All rights reserved.