caa a22 4500 605499004 CHVBK 20210128100549.0 cr unu---uuuuu 210128e20150301xx s 000 0 eng 10.1007/s00253-014-6061-4 doi (NATIONALLICENCE)springer-10.1007/s00253-014-6061-4 Improvement of the amides forming capacity of the arylacetonitrilase from Pseudomonas fluorescens EBC191 by site-directed mutagenesis [Elektronische Daten] [Olga Sosedov, Andreas Stolz] The influence of different amino acid substitutions in the nitrilase from Pseudomonas fluorescens EBC191 (NitA) on the catalytical activity and the ability to form amides was investigated. The enzyme variant Glu137Ala was constructed because glutamate residues homologous to Glu137 are highly conserved among different members of the nitrilase superfamily and it has been suggested that these residues are indispensable for the hydrolysis of amides by enzymes belonging to the nitrilase superfamily. The enzyme variant Glu137Ala demonstrated less than 1% of the wild-type activity but was still enzymatically competent to convert mandelonitrile to mandelic acid and mandeloamide. The tryptophan residue at position 188, which was previously identified as important for the amide forming capacity of the nitrilase, was exchanged by saturation mutagenesis for all other proteinogenic amino acids. Surprisingly, 18 of these 19 exchanges resulted in an increased formation of mandeloamide from (R,S)-mandelonitrile and three of these variants converted (R,S)-mandelonitrile to more than 90% of mandeloamide. Furthermore, these modifications also resulted in a reversal of stereoselectivity and these variants formed in contrast to the wild-type enzyme and almost all other known nitrilases preferentially (S)-mandelic acid. The synthetic potential of one of these variants was demonstrated by the construction of recombinant E. coli clones which simultaneously expressed the nitrilase variant and the (S)-hydroxynitrile lyase (oxynitrilase) from the cassava plant (Manihot esculenta). These "bienzymatic catalysts” converted benzaldehyde plus cyanide almost exclusively to (S)-mandeloamide and did not show any inhibition in the presence of cyanide in concentrations up to 200mM. Springer-Verlag Berlin Heidelberg, 2014 Nitrilase nationallicence Nitrile hydratase nationallicence Oxynitrilase nationallicence Mandelic acid nationallicence Cascade reactions nationallicence Sosedov Olga Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, 70569, Stuttgart, Germany aut Stolz Andreas Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, 70569, Stuttgart, Germany aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/6(2015-03-01), 2623-2635 0175-7598 99:6<2623 2015 99 253 https://doi.org/10.1007/s00253-014-6061-4 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-6061-4 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Sosedov Olga Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, 70569, Stuttgart, Germany aut NATIONALLICENCE 700 1- Stolz Andreas Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, 70569, Stuttgart, Germany aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/6(2015-03-01), 2623-2635 0175-7598 99:6<2623 2015 99 253