Penicillin acylase activity of LapBSH

The penicillin acylase activity of purified LapBSH protein was demonstrated by gas chromatograph mass spectrometry (GC-MS) analysis as described previously (Kusada et al., 2017 (link); Yasutake et al., 2017 (link)). Briefly, 10 mM of penicillin G (Nacalai Tesque) solution was mixed with the purified LapBSH and buffer as a no-enzyme control and incubated at 37°C. After incubation, the digestion mixtures were extracted three times with equal volumes of ethyl acetate (FUJIFILM Wako Pure Chemical Corporation). Thereafter, the organic phase was evaporated to dryness in vacuum for 10 min (EYELA, Tokyo, Japan). The resulting sample was re-dissolved in methanol (FUJIFILM Wako Pure Chemical Corporation) and introduced onto a SHIMADZU GCMS-QP5050 system (Shimadzu Co., Ltd., Kyoto, Japan) equipped with a DB-5 capillary column (30 m × 0.25 mm, 0.25-μm film thickness; Agilent Technologies, Palo Alto, CA, United States).