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Affinity chromatographic separation of secreted alkaline phosphatase and glucoamylase using reactive dyes

Paper ID Volume ID Publish Year Pages File Format Full-Text
36010 45118 2007 9 PDF Available
Title
Affinity chromatographic separation of secreted alkaline phosphatase and glucoamylase using reactive dyes
Abstract

A single-step dye affinity chromatographic separation method was developed to separate secreted alkaline phosphatase (SEAP) and glucoamylase produced in CHO cell culture and Aspergillus niger fermentation, respectively. The reactive dye, Procion® Green H-E4BD, was found to have a good binding capacity for SEAP, whereas Procion® Blue H-ERD was the best dye ligand for glucoamylase. However, these dyes have a relatively low selectivity for the target protein. Consequently, elution of the adsorbed proteins by KCl solution resulted in a product with many impurity proteins as evident by the multiple protein bands on SDS-PAGE. However, elution of SEAP by its substrate, phosphate, produced a relatively pure protein with a high specific enzyme activity because of the competition for active site between the substrate and the dye ligand. Also, a high-purity glucoamylase product was obtained by elution with a borate solution. The relatively inexpensive dye affinity chromatography thus can be used for purifying enzymes from cell culture and fermentation broths. The adsorption of SEAP on the dye-ligand affinity resin followed the Langmuir isotherm. An axial dispersion model with external mass transfer limitation was developed to simulate the breakthrough curve in the chromatographic column. This mathematical model can be used to scale up the protein adsorption process.

Keywords
Affinity chromatography; Reactive dye; Secreted alkaline phosphatase (SEAP); Glucoamylase; Separation
First Page Preview
Affinity chromatographic separation of secreted alkaline phosphatase and glucoamylase using reactive dyes
Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 42, Issue 4, April 2007, Pages 561–569
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering