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Site directed immobilization of glucose-6-phosphate dehydrogenase via thiol-disulfide interchange: Influence on catalytic activity of cysteines introduced at different positions

Paper ID Volume ID Publish Year Pages File Format Full-Text
23544 43451 2013 7 PDF Available
Title
Site directed immobilization of glucose-6-phosphate dehydrogenase via thiol-disulfide interchange: Influence on catalytic activity of cysteines introduced at different positions
Abstract

•Site specific immobilization of G6PDH mutants G6PDH D205C, G6PDH L218C and G6PDH D453C.•Free G6PDH D205C and G6PDH D453C showed comparable enzyme activities to the G6PDH wt.•Immobilized G6PDH L218C exhibited a protein specific activity loss of at least 50%.•Immobilized G6PDH D205C showed a minor activity reduction by immobilization.•However, immobilized G6PDH D453C exhibited a recovery in enzymatic activity of 100%.

This study shows the effect of site-directed enzyme immobilization upon the enzyme activity of covalently bound glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Immobilization points were introduced at sterically accessible sites in order to control the protein's orientation and twice as much activity was recovered in comparison to conventionally immobilized enzyme. Immobilization of G6PDH via genetically engineered cysteine provided a simple, but effective method to control the immobilization process. G6PDH variants with cysteine close to the active center (L218C), close to the dimer interface (D205C) as well as far from the active center (D453C) showed changes in activity and the efficacy of immobilization.

Keywords
2-PDS, 2-pyridyldisulfide; A343, absorption at 343 nm; DTT, dithiothreitol; G6P, glucose-6-phosphate; G6PDH, glucose-6-phosphate dehydrogenase; IPTG, isopropyl-β-d-thiogalactopyranosid; LB, lysogenic broth; NADP+, nicotinamide adenine dinucleotide phospha
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Site directed immobilization of glucose-6-phosphate dehydrogenase via thiol-disulfide interchange: Influence on catalytic activity of cysteines introduced at different positions
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 167, Issue 1, 10 August 2013, Pages 1–7
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