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The co-operative effect of physical and covalent protein adsorption on heterofunctional supports

Paper ID Volume ID Publish Year Pages File Format Full-Text
35135 45077 2009 7 PDF Available
Title
The co-operative effect of physical and covalent protein adsorption on heterofunctional supports
Abstract

It has been found that the enzymes penicillin G acylase from Escherichia coli (PGA) and lipase from Bacillus thermocatenulatus (BTL) did not significantly adsorb on highly activated amino-agarose beads at pH 7 (a support where 85–90% of a crude extract of proteins become adsorbed). Moreover, it has been found that these enzymes do not covalently immobilize on highly activated epoxy-agarose beads at pH 7. However, both enzymes slowly immobilize on heterofunctional supports having a high density of amino–epoxy groups. The immobilized enzymes retain a high percentage of activity (more than 90% for PGA and 60% for BTL). On the other hand, the immobilization of a crude extract of proteins on amino–epoxy supports under conditions where only a limited protein ionic exchange was permitted (by using high ionic strength or lowly activated supports), also permitted a similar high immobilization yield of the proteins. Similarly, glutamate dehydrogenase (GDH) and β-galactosidase from Thermus thermophilus can be fully immobilized under conditions where less than 20% of these enzymes can be ionically exchanged in the aminated support. The results suggested that the percentage of proteins that may be physically adsorbed on the support becomes irreversibly immobilized by the covalent reaction between the nucleophilic groups in the protein surface and the very near epoxy groups of the support (in an almost intramolecular reaction). Thus, using these supports, it is possible to immobilize almost all the proteins by anionic exchange, that is, the area with the highest density in anionic groups. In many cases, this region could not correspond to the protein regions usually utilized to immobilize proteins. This way, it is possible to achieve, in a very simple fashion and without modifying the protein, new orientations of some immobilized enzymes and proteins.

Keywords
Immobilization of proteins on epoxy supports; Amino–epoxy supports; Intramolecular covalent attachment; Intermolecular covalent attachment; Two step immobilization; Oriented immobilization
First Page Preview
The co-operative effect of physical and covalent protein adsorption on heterofunctional supports
Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 44, Issue 7, July 2009, Pages 757–763
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering