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 His-tag 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 K (m) 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.
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