Biochemical characterization of an alcohol dehydrogenase from Ralstonia sp.
Article first published online: 22 FEB 2013
Copyright © 2013 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 110, Issue 7, pages 1838–1848, July 2013
How to Cite
Kulig, J., Frese, A., Kroutil, W., Pohl, M. and Rother, D. (2013), Biochemical characterization of an alcohol dehydrogenase from Ralstonia sp. Biotechnol. Bioeng., 110: 1838–1848. doi: 10.1002/bit.24857
- Issue published online: 25 MAY 2013
- Article first published online: 22 FEB 2013
- Accepted manuscript online: 4 FEB 2013 08:02AM EST
- Manuscript Accepted: 22 JAN 2013
- Manuscript Revised: 21 DEC 2012
- Manuscript Received: 4 SEP 2012
- Marie Curie Initial Training Network in frame of project “BIOTRAINS—a European biotechnology training network for the support of chemical manufacturing”. Grant Number: 238531
- alcohol dehydrogenase;
- Ralstonia sp.;
- Cupriavidus sp.;
- calcium dependency;
- bulky substrates
Stereoselective reduction towards pharmaceutically potent products with multi-chiral centers is an ongoing hot topic, but up to now catalysts for reductions of bulky aromatic substrates are rare. The NADPH-dependent alcohol dehydrogenase from Ralstonia sp. (RADH) is an exception as it prefers sterically demanding substrates. Recent studies with this enzyme indicated outstanding potential for the reduction of various alpha-hydroxy ketones, but were performed with crude cell extract, which hampered its detailed characterization. We have established a procedure for the purification and storage of RADH and found a significantly stabilizing effect by addition of CaCl2. Detailed analysis of the pH-dependent activity and stability yielded a broad pH-optimum (pH 6–9.5) for the reduction reaction and a sharp optimum of pH 10–11.5 for the oxidation reaction. The enzyme exhibits highest stability at pH 5.5–8 and 8–15°C; nevertheless, biotransformations can also be carried out at 25°C (half-life 80 h). Under optimized reaction parameters a thorough study of the substrate range of RADH including the reduction of different aldehydes and ketones and the oxidation of a broad range of alcohols was conducted. In contrast to most other known alcohol dehydrogenases, RADH clearly prefers aromatic and cyclic aliphatic compounds, which makes this enzyme unique for conversion of space demanding substrates. Further, reductions are catalyzed with extremely high stereoselectivity (>99% enantio- and diastereomeric excess). In order to identify appropriate substrate and cofactor concentrations for biotransformations, kinetic parameters were determined for NADP(H) and selected substrates. Among these, we studied the reduction of both enantiomers of 2-hydroxypropiophenone in more detail. Biotechnol. Bioeng. 2013; 110: 1838–1848. © 2013 Wiley Periodicals, Inc.