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Production and separation of formate dehydrogenase from Candida boidinii

Paper ID Volume ID Publish Year Pages File Format Full-Text
18060 42710 2007 7 PDF Available
Title
Production and separation of formate dehydrogenase from Candida boidinii
Abstract

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 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, ∼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.

Keywords
Formate dehydrogenase (FDH); Dye affinity chromatograph; Polyethyleneimine (PEI); Candida boidinii
First Page Preview
Production and separation of formate dehydrogenase from Candida boidinii
Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 40, Issue 4, 5 March 2007, Pages 940–946
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering