Sunday, July 26, 2009
P39

Improved nitrilase activity at low pH

Ulrike Schreiner1, Bianca Hecher1, Sascha Obrowsky1, Kerstin Waich1, Margit Winkler1, David Rozzell2, and Anton Glieder1. (1) Applied Biocatalysis Research Centre, Petersgasse 14/3, Graz, Austria, (2) Codexis Inc., 200 Penobscot Drive, Redwood City, CA 94063

Nitrilases catalyze the hydrolysis of nitriles to their corresponding carboxylic acids and ammonia. They exhibit their optimal activity at neutral pH values. For hydrolysis of base labile substrates such as cyanohydrins we aspired to improve the stability of the Alcaligenes faecalis nitrilase (NITAf, swissprot: P20960) for the hydrolysis of cyanohydrins at low pH by directed evolution. Mutant pHNIT45 showed a 3 to 4 fold higher specific activity compared to NITAf upon incubation at pH 4.5 for 30 minutes. To prove the applicability of the newly engineered pHNIT45 we studied the conversion of (R)-2-Cl-mandelonitrile to (R)-2-Cl-mandelic acid at pH 4.5 and pH 7.5 and compared it to NITAf. At pH 4.5 (R)-2-Cl-mandelic acid was obtained with improved conversion and higher enantiopurity than NITAf at pH 7.5.


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