US EPA

1228105 

Journal Article 

Production and separation of formate dehydrogenase from Candida boidinii 

Bai, Y; Yang, ST 

2007 

Yes 

Enzyme and Microbial Technology
ISSN: 0141-0229 

Elsevier Science Ltd., The Boulevard Langford Lane Kidlington Oxford OX5 1GB UK, [mailto:nlinfo-f@elsevier.nl], [URL:http://www.elsevier.nl] 

40 

4 (Mar 2007) 

940-946 

10.1016/j.enzmictec.2006.07.035 

WOS:000245015600060 

The production of FDH in a 40-l bioreactor was carried out with methanol as the inducer to reach the final intracellular FDH activity of 35U/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 0.04mg/ml to the cell extract, 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 5mM NAD+ in 0.2M KCl improved the FDH elution and increased the specific FDH activity by 1.38-fold as compared to elution with 1M 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. 

Affinity chromatography; Enzymes; Cell walls; potassium chloride; Impurities; Herbs; Methanol; polyethyleneimine; Bioreactors; Formate dehydrogenase; Candida boidinii; Precipitation; Toluene