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Horse Liver Alcohol Dehydrogenase: new perspectives for an old enzyme
Date Issued
2012-11
Date Available
2013-11-01T04:00:08Z
Abstract
The EE subunit of horse liver alcohol dehydrogenase
(HLADH-EE) has been subcloned in pRSETb vector
to generate a fusion His-tag protein. The migration from a
multistep purification protocol for this well-known enzyme
to a single-step has been successfully achieved. Several
adjustments to the traditional purification procedure for Histag
proteins have been made to retain protein activity. A full
characterization of the fusion enzyme has been carried out
and compared with the native one. The Km for EtOH, NAD
and NADH in the His-tag version of HLADH are in line
with the ones reported in literature for the native enzyme. A
shift in optimal pH activity is also observed. The enzyme
retains the same stability and quaternary structure as the
wild type and can therefore be easily used instead of the
native HLADH for biotechnological applications.
(HLADH-EE) has been subcloned in pRSETb vector
to generate a fusion His-tag protein. The migration from a
multistep purification protocol for this well-known enzyme
to a single-step has been successfully achieved. Several
adjustments to the traditional purification procedure for Histag
proteins have been made to retain protein activity. A full
characterization of the fusion enzyme has been carried out
and compared with the native one. The Km for EtOH, NAD
and NADH in the His-tag version of HLADH are in line
with the ones reported in literature for the native enzyme. A
shift in optimal pH activity is also observed. The enzyme
retains the same stability and quaternary structure as the
wild type and can therefore be easily used instead of the
native HLADH for biotechnological applications.
Type of Material
Journal Article
Publisher
Springer Science+Business Media
Journal
Molecular Biotechnology
Volume
52
Issue
3
Start Page
244
End Page
250
Copyright (Published Version)
2012 Springer Science+Business Media, LLC
Subject – LCSH
Chromatographic analysis
Alcohol dehydrogenase
Enzymes--Analysis
Language
English
Status of Item
Not peer reviewed
This item is made available under a Creative Commons License
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Name
Quaglia_J_Biotech_2012.doc
Size
304 KB
Format
Microsoft Word
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