P. putida KT2400-tacRDL was inoculated from the overnight cultivated seeds into 50 mL LB containing 25 mg/L kanamycin, and cultivated at 37 °C and 220 rpm for 24 h. This expression system is constitutive due to the absence of lacI both in the used plasmid and P. putida KT2400 [23 (link),41 (link)], so it does not need the addition of isopropyl β-d-1-thiogalactopyranoside (IPTG). Cells were removed by centrifugation at 12,000 rpm and the cell-free supernatant was filtered with a 10 kDa ultrafilter (Millipore, Burlington, MA, USA) and concentrated to obtain 1 mL LCC crude enzyme. Cells were disrupted with an automatic sample rapid grinder (Jingxin Technology, Shanghai, China) [42 (link)] and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), as with the cell-free supernatant, before and after concentration. Total protein concentration of the crude enzyme was determined with the folin phenol reagent [36 (link)], and the content of LCC was determined by image analysis using online ImageJ [37 (link)] (https://cnij.imjoy.io/ (accessed on 24 February 2022)).
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