B per complete reagent

For in vivo characterization of CAT_Sa and its variants in E. coli, high-cell density cultures were performed as previously described [53 (link)] with an addition of 2 g/L of various alcohols. For in situ extraction of esters, each tube was overlaid with 25% (v/v) hexadecane. To confirm the protein expression of CAT_Sa and its variants, 1% (v/v) of stock cells were grown overnight at 37 °C and 200 rpm in 15 mL culture tubes containing 5 mL of LB medium and antibiotic. Then, 4% (v/v) of the overnight cultures were transferred into 1 mL of LB medium containing antibiotic in a 24-well microplate. The cultures were grown at 37 °C and 350 rpm using an incubating microplate shaker (Fisher Scientific, PA, USA) until OD reached to 0.4–0.6 and then induced by 0.1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 4 h with a Breathe-Easy Sealing Membrane to prevent evaporation and cross contamination (cat# 50-550-304, Research Products International Corp., IL, USA). The protein samples were obtained using the B-PER complete reagent (cat# 89822, Thermo Scientific, MA, USA), according to the manufacturer’s instruction and analyzed by SDS-PAGE.

BsADH2 was cultivated at 37 ​°C/225 ​rpm for 8 ​h after induction. Then 10 ​mL of the cell culture was centrifuged at 20,000 ​g for 10 ​min. The supernatant was decanted. The cell pellets were frozen at −20 ​°C for 2 ​h. Subsequently, 1 ​mL of B-PER Complete Reagent (Thermo Scientific) was added to resuspend the cells. After being incubated at room temperature for 15 ​min, the cell lysates were centrifuged at 16,000 ​g for 20 ​min. The supernatant was used for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) based on the manufacturer’s instruction.
In the case of the BsADH1, the cells were cultured in 10 ​mL of the aerobic medium with 60 ​g/L of glucose in a 20-mL serum bottle with a butyl stopper. After being incubated at 37 ​°C for 8 ​h, the cell culture was centrifuged at 20,000 ​g for 10 ​min. Then the cell pellets were frozen at −20 ​°C for 2 ​h and lysed by B-PER. After the cells were lysed, the supernatant was used for SDS-PAGE. A control (Bs1A1) was also included.