Biotransformation of nitriles and amides
Organic nitriles of the general structure "R-CN" are easily accessible synthetically and are widely used in the chemical industry as building blocks for further syntheses. Therefore, within the scope of our investigations, we are looking for microorganisms or enzymes that convert easy-to-synthesise nitriles by chemo-, regio- and enantioselective reactions into higher-value industrially relevant products. Enzymatic nitrile hydrolysis is carried out either by nitrile hydratases to the corresponding amides or by nitrilases to the carboxylic acids. Thus, both optically active carboxylic acids and carboxamides are accessible by enantioselective enzymatic hydrolysis.
In the course of our investigations, numerous enrichments were carried out in which different nitriles or amides were offered as the sole nitrogen source in the presence of an energy-providing auxiliary substrate. It was shown that the selection principle used opens up a variety of novel nitrile-saponifying biological systems (Layh et al. 1997; Rustler et al., 2007). In these studies, the focus was initially placed on the synthesis of optically pure alpha-arylpropionic acids, which, as so-called profenes (e.g. ibuprofen, ketoprofen, naproxen),have great commercial importance as non-steroidal anti-inflammatory agents. In the case of profenes, only one of the enantiomers has the pharmacological effect. In these studies, it was possible to isolate a bacterial strain that produced almost exclusively the desired enantiomer of profenes from a racemic precursor with the aid of a nitrile hydratase/amidase system (Layh et al., 1992, 1994, 1995; Bauer et al., 1994).
In further work, the amidase responsible for the enantioselective reaction was purified and characterised, and the coding gene was characterised molecularly and efficient expression systems were constructed (Hirrlinger et al., 1996; Trott et al., 2002). In further work, in collaboration with the Institute of Organic Chemistry at the Technical University of Delft, it was shown that related nitrile hydratase-amidase systems can also be used to produce optically active amino acid amides from aminonitriles (Wegman et al., 2000, 2001).In recent years, more investigations have been carried out with bacterial nitrilases. A number of nitrilases from different bacterial strains have been cloned (Heinemann et al., 2003a,b; Kiziak et al., 2005; Kaul et al., 2007)and, using the example of a nitrilase from Pseudomonas fluorescens, analysed the molecular basis for the chemo- and enantiospecificity of bacterial nitrilases by producing various mutants (Kiziak et al., 2005, 2007; Kiziak & Stolz, 2009; Fernandes et al., 2006).
Within the framework of a joint project with the TU Delft, in vitro and in vivo coupled enzyme systems from a plant oxynitrilase and the nitrilase from P. fluorescens were developed and used for the synthesis of optically active hydroxycarboxylic acids (Mateo et al., 2006; Sosedov et al., 2009, 2010).
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