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Heterofunctional supports for the one-step purification, immobilization and stabilization of large multimeric enzymes: Amino-glyoxyl versus amino-epoxy supports

Paper ID Volume ID Publish Year Pages File Format Full-Text
35514 45093 2010 7 PDF Available
Title
Heterofunctional supports for the one-step purification, immobilization and stabilization of large multimeric enzymes: Amino-glyoxyl versus amino-epoxy supports
Abstract

For the immobilization-stabilization of multimeric enzymes, we propose a novel heterofunctional support containing a very low concentration of ionized amino groups and a very high concentration of very poorly reactive glyoxyl (aldehyde) groups. A large tetrameric enzyme, β-galactosidase from Thermus sp., was purified and dramatically stabilized with this novel support. The enzyme was first immobilized by physical adsorption via selective multipoint anionic exchange involving the largest region of the enzyme containing all enzyme subunits. Then, an additional long incubation of the immobilized derivative under alkaline conditions was performed in order to promote an intense intramolecular multipoint covalent attachment between amino groups of the adsorbed enzyme and the very stable glyoxyl groups on the support. This novel β-galactosidase derivative is the first one in which the four subunits of this enzyme become attached to a pre-existing support. Additionally, the novel amino-glyoxyl supports were much more suitable than amino-epoxy supports for intramolecular multipoint covalent immobilization of the adsorbed enzyme onto the support. In fact, at pH 7.0, the new supports covalently immobilize the physically adsorbed protein 24-fold more rapidly than epoxy supports. Furthermore, derivatives prepared on amino-glyoxyl supports preserved 85% of catalytic activity and were 5-fold more stable than derivatives prepared on amino-epoxy supports and more than 1000-fold more stable than soluble enzyme.

Keywords
Intramolecular covalent attachment of adsorbed enzymes; Stabilization of multimeric enzymes; Selective immobilization of very large proteins
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Heterofunctional supports for the one-step purification, immobilization and stabilization of large multimeric enzymes: Amino-glyoxyl versus amino-epoxy supports
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 45, Issue 10, October 2010, Pages 1692–1698
Authors
, , , , , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us