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Promotion of multipoint covalent immobilization through different regions of genetically modified penicillin G acylase from E. coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
35118 45076 2010 9 PDF Available
Title
Promotion of multipoint covalent immobilization through different regions of genetically modified penicillin G acylase from E. coli
Abstract

A novel approach is proposed to prepare a set of immobilized derivatives of a enzyme covalently rigidified through different regions of its surface. Six different variants of penicillin G acylase (PGA) from Escherichia coli (which lacks Cys) were prepared by introducing a unique Cys residue via site-directed mutagenesis in six different enzyme regions which were rich in Lys residues. All variants exhibited a similar activity and stability compared to those of the native enzyme. Each variant was immobilized on supports having a low concentration of reactive disulfide moieties and a high concentration of poorly reactive epoxy groups. After immobilization at pH 7.0 by site-directed thiol-disulfide intermolecular exchange, derivatives were further incubated at pH 10.0 for 48 h to promote an additional intramolecular reaction between Lys residues of enzyme and epoxy groups of the support. The establishment of at least three covalent attachments per PGA molecule was determined for all immobilized enzyme variants. The different derivatives exhibited diverse stability against several distorting agents and different selectivity in two interesting reactions. The derivative of the PGA variant obtained by replacement of GlnB380 by Cys was the most stable against heat and organic cosolvents: it preserved 90% of the initial activity and was 30-fold more stable than soluble PGA. This derivative also exhibited an improved enantioselectivity in the hydrolysis of chiral esters (E was improved from 8 to 16) and in kinetically controlled synthesis of amides (synthetic yields were increased from 31 to 49%).

Keywords
Directed immobilization; Multipoint covalent attachment; Directed rigidification; Enzyme stabilization; Enzyme selectivity; Thiol-epoxy supports
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Promotion of multipoint covalent immobilization through different regions of genetically modified penicillin G acylase from E. coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 45, Issue 3, March 2010, Pages 390–398
Authors
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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