398177 E. coli Pilot-Scale One-Step Purification of Amine Dehydrogenases Employing an Aqueous Two-Phase Extraction System

Monday, November 17, 2014
Galleria Exhibit Hall (Hilton Atlanta)
Junxian Wu1, Bettina Bommarius2, Samantha Au3 and Andreas S. Bommarius1, (1)Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, (2)School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, (3)Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA

This project explores for the first time the use of a large-scale aqueous two-phase extraction system (ATPS) to purify dehydrogenases. Dehydrogenases are an important family of enzymes that generates chiral alcohols and amines for pharmaceutical and crop protection agents. This work first explores the purification of popular dehydrogenases (GDH, FDH) as well as the newly developed amine dehydrogenase family (L-AmDH, F-AmDH and cFL1-AmDH) in an ATPS. In aqueous two-phase systems (ATPS), when polymers and salts are added to water, the water forms two phases.  The ATPS consists of a top polymer layer and a bottom salt layer. The goal is to selectively partition the dehydrogenase into the top phase to achieve pure protein, while the cell lysate and debris will remain in the bottom phase. Each enzyme will require different purification conditions (PEG, salt, temperature, etc.) and this work investigates these conditions and develops a general purification protocol.

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