Mandelonitrile

For determination of substrate specificity, several nitriles belonging to different classes were tested: acetonitrile (simple aliphatic nitrile), glutaronitrile, succinonitrile (aliphatic dinitriles), mandelonitrile (arylacetonitrile) and benzonitrile (aromatic nitrile) (Sigma-Aldrich). In addition, acetamide and benzamide (Sigma-Aldrich) were used for the detection of possible amidase activity.

Both cell-based and CFPS preparations were assayed to compare putative nitrilase activity on representative substrates. Benzonitrile, cinnamonitrile, 3-phenylpropionitrile, mandelonitrile, and 2-thiopheneacetonitrile were all obtained from Sigma Aldrich. Reactions were assayed under the following conditions: 55 µL of 100 mM KPi buffer pH 7.0, 5 µL of 400 mM nitrilase substrate in DMSO, 40 μL of lysate (either CFPS or cell-based as described above), total reaction volume (100 µL in 96-well Nunc Flat plate). The reaction plates were sealed with a pierceable aluminum seal and incubated for approximately 15 hours at 30 °C and 500 rpm. In order to prepare the plates for analysis, the reactions were diluted with 100 µL of acetonitrile and then sealed and shaken for 40 min at 900 rpm at room temperature. The solid debris was then pelleted via centrifugation at 4000 rpm for 10 min at 4 °C. The clarified supernatant was then diluted 10x into 50:50 water:acetonitrile prior to HPLC analysis. Peak areas were integrated at 230 nm for the nitrilase substrate and hydrolysis product (except for samples containing 3-phenylpropionitrile which were integrated at 210 nm) and nitrilase hydrolysis activity was determined by: peak area hydrolysis product/(peak area hydrolysis product + peak area nitrile) × 100% (see Supplementary Fig. S3 and representative chromatograms in Supplementary Methods).

Stock solutions of nitriles (final concentration of 2.7 M) were generated by solving the nitriles in N,N-dimethylformamide (DMF) (Merck KGaA, Darmstadt, Germany). These stock solutions were sterile-filtered and stored at 4 °C. The following nitriles were used: phenylacetonitrile, acetonitrile (both TCI Deutschland GmbH, Eschborn, Germany), succinonitrile, crotonitrile, 4-hydroxybenzonitrile, acetone cyanohydrin, cyclohexanecarbonitrile, fumaronitrile, and 2-phenylpropionitrile (all Sigma-Aldrich Chemie GmbH, Munich, Germany). In addition, mandelonitrile, 2-phenylbutyronitrile, benzonitrile, 3-indoleacetonitrile, 2-thiopheneacetonitrile, 3-thiopheneacetonitrile, (2-chlorophenyl)acetonitrile, (3-chlorophenyl)acetonitrile, (4-chlorophenyl)acetonitrile, cinnamonitrile, 1,4-phenylenediacetonitrile, 2-naphtylacetonitrile (all Sigma-Aldrich), and 3-phenylpropiontrile (Alfa Aesar, Haverhill, MA, USA) were used for further characterization of the purified arylacetonitrilase.