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Stabilization of a highly active but unstable alcohol dehydrogenase from yeast using immobilization and post-immobilization techniques

Paper ID Volume ID Publish Year Pages File Format Full-Text
34559 45034 2012 8 PDF Available
Title
Stabilization of a highly active but unstable alcohol dehydrogenase from yeast using immobilization and post-immobilization techniques
Abstract

The alcohol dehydrogenase (ADH) from Baker's yeast is very active but extremely unstable under several different conditions. Mild immobilization methods such as one-point attachment to agarose activated with cyanogen bromide groups or ionic adsorption to agarose activated with charged groups allow high activity recoveries (80–100%) but do not promote protein stabilization. In contrast, immobilization methods that force the enzyme to be covalently attached at multiple points on the support fully inactivate the enzyme. Herein, we propose an interesting solution to address the dichotomy between activity and stability. We have developed a protocol in which the enzyme is immobilized on agarose activated with glyoxyl groups in the presence of acetyl cysteine, which results in the recovery of 25% of the enzyme activity but increases the thermal stability of the soluble enzyme 50-fold. However, this immobilization technique does not stabilize the enzyme quaternary structure. Hence, a post-immobilization technique using functionalized polymers has been used to cross-link all enzyme subunits. In this method, polycationic polymers (polyethylenimine) cross-link the quaternary structure with a negligible effect on catalytic activity, which results in a derivative that is 5-fold more stable than non-cross-linked derivatives under very dilute and acidic conditions that highly favor subunit dissociation. Therefore, the stability was increased 500-fold for this optimal derivative compared to diluted soluble enzyme, although the relative expressed activity was low (25%). However, the low expressed activity may be overcome by designing immobilized biocatalysts with high volumetric activities.

► A multidisciplinary approach to greatly stabilize a very relevant biotechnological enzyme. ► The major stabilization of alcohol dehydrogenase from yeast ever reported. ► Chemical and physical approaches for enzyme stabilization. ► Stabilization of a multimeric enzyme by physical crosslinking of subunits.

Keywords
Protein stabilization; Post-immobilization techniques; Alcohol dehydrogenase; Multimeric enzymes; Asymmetric chemistry
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Stabilization of a highly active but unstable alcohol dehydrogenase from yeast using immobilization and post-immobilization techniques
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 47, Issue 5, May 2012, Pages 679–686
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