4.7. Enzymatic Synthesis of Penicillin V and AHLs

Kinetically controlled synthesis of penicillin V by AuAHLA was carried out according to Hormigo et al. [36] with slight modifications. The reaction mixture containing 1 IU/mL AuAHLA, 100 mM 6-APA, 20 mM MPOA and 3% or 32% (v/v) DMSO, was incubated in 100 mM potassium phosphate pH 7.0 buffer at 30 °C and 800 rpm. At different incubation times, 20 µL of the enzymatic reaction were mixed with 20 µL of cold methanol and analyzed by HPLC using an Agilent 1100 equipment (Santa Clara, CA, USA) and a Phenomenex (Torrance, CA, USA) Luna column C18(2), 250 × 4.6 mm (5 μM particle size); with a 65% (v/v) methanol solution containing 0.05% (v/v) phosphoric acid as mobile phase. The flow rate was fixed at 0.8 mL/min, and the UV detector was set at 260 nm. Retention times of substrates and products were 4.0 (6-APA), 6.6 (POA), 9.6 (MPOA), and 11.3 min (penicillin V). Concentrations of penicillin V, POA, and MPOA were estimated with calibration curves using standard solutions. All determinations were carried out in triplicate. Synthetic activity (V_S) was established as the initial rate of synthesis (penicillin V production per unit time, expressed as mM/h), whereas hydrolytic activity (V_h) was established as the initial rate of hydrolysis of MPOA (POA production per unit time, expressed as mM/h). The ratio of synthetic activity to hydrolytic activity (S/H ratio) was calculated as V_S/V_h.

Enzymatic synthesis of AHLs were carried out with 2 IU/mL AuAHLA, 100 mM L-HSL, 20 mM acyl donor (methyl butyrate, hexanoate, octanoate, decanoate, dodecanoate, butyrylacetate, 3-oxodecanoate, or 3-oxododecanoate), and 23% or 50% (v/v) DMSO in 100 mM potassium phosphate pH 7.0 buffer. These mixtures were incubated at 30 °C and 800 rpm for 24 h, and then the samples were evaporated at 45 °C to dryness using a speed vacuum concentrator Thermo Scientific SPD121P (Waltham, MA, USA), stored at −20 °C, and dissolved in methanol prior to analysis. Enzymatic synthesized AHLs were analyzed by LC-MS/MS using a Shimadzu LCMS8030 (Kyoto, Japan) equipment and a Phenomenex (Torrance, CA, USA) Gemini Column C18 110 Å (150 × 2 mm 5 μm particle size) with a Phenomenex C18 pre-column (4 × 2 mm) in the Mass Spectrometry Facility (Faculty of Chemistry, UCM). Mobile phase A was water and mobile phase B was methanol, and the gradient profile was as follows: 0% phase B for 1 min, from 0% to 100% phase B for 5 min, 100% phase B for 1.5 min, from 100% to 0% phase B for 1 min and 0% phase B for 1.5 min at a flow rate of 0.4 mL/min. Analysis of AHLs was performed by multiple reaction monitoring (MRM) mode to monitor de parent ion-product ion (m/z) of the analyte, and mass transitions of each molecule are listed in Supplementary Material, Table S3.