Ultrasonication

Cells stimulated with methanol were collected at various time points during their incubation, concentrated by centrifugation at 12,000× g at 4 °C, and then disrupted by ultrasonication (Thermo Fisher Scientific, Waltham, MA, USA). Proteins in the supernatant were precipitated with trichloroacetic acid, boiled for 10 min and mixed with an equal volume of 1× loading buffer. SDS-PAGE was performed on a 10% polyacrylamide gel and Coomassie Brilliant Blue R250 was used to detect the protein bands. After transfer onto a polyvinylidene fluoride membrane, CarE was detected with an anti-His mouse polyclonal antibody (Cell Signalling Technology, Danvers, UT, USA) and diaminobenzidine substrate (Shenggong, Shanghai, China).

The recombinant cells were harvested and resuspended in buffer A (50 mM sodium monophosphate, 300 mM NaCl, 10 mM imidazole, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF) as a protease inhibitor). The resuspended cells were disrupted by ultrasonication (Fisher Scientific, Pittsburgh, PA) on ice. The cell debris was removed by centrifugation at 15,000 × g for 20 min at 4°C and the supernatant was filtered through a 0.45 μm filter and applied to an IMAC chromatography column (Bio-Rad, Hercules, CA) equilibrated with buffer A. The bound protein was eluted with a linear gradient between 10 mM and 250 mM imidazole in buffer A. The active fraction was dialyzed in 50 mM HEPES buffer (pH 7.5) and the resulting solution was used as the purified enzyme. All purification steps using columns were carried out using a Profinia^™ Affinity Chromatography Protein Purification System (Bio-Rad). The protein concentration was quantified by the method reported by Bradford[11 (link)]. The purified proteins were confirmed by SDS-PAGE gel analysis.

The pTrc-nitAF-transformed, overnight-grown BL21 (DE3) cells (Invitrogen, Carlsbad, CA, USA) were diluted 200-fold in LB with ampicillin (100 µg/mL) and grown at 30 °C until the suspension attained an OD₆₀₀ of ~0.6. IPTG (0.1 mM) was used to induce protein expression for 3 h of incubation. Prior to cell disruption through ultrasonication (Fisher Scientific, Pittsburgh, PA, USA), cells were treated with lysozyme (1 mg/mL) in potassium phosphate buffer (50 mM) containing 0.1 mM each of dithiothreitol, EDTA, and phenylmethyl-sulfonyl fluoride as protease inhibitors. After removing cell debris, the supernatant loaded into the Ni-NTA column (3.4 × 13.5 cm, QIAGEN) was pre-equilibrated using lysis buffer (NaH₂PO₄, 50 mM; NaCl, 300 mM; pH 8). Further, free proteins were separated using washing buffer (NaH₂PO₄, 50 mM; NaCl, 300 mM; imidazole, 20 mM; pH 8). The bound arylacetonitrilase was recovered using elution buffer (NaH₂PO₄, 50 mM, NaCl, 300 mM; imidazole, 250 mM; pH 8). Similarly, recombinant nitrilases (tagged with histidine residues at the C terminal) were purified using the Ni-NTA system. Protein quantification and purity confirmation were evaluated by Bradford [30 (link)] and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) methods, respectively.