Penicillin Acylase Activity Assay

The penicillin acylase activity of the purified protein was determined as described previously with slight modifications (Kusada et al., 2017 (link); Yasutake et al., 2017 (link)). Briefly, the purified LpBSH protein was mixed with 10 mM penicillin G solution and incubated at 37°C. As a negative control, penicillin G solution was mixed with a buffer (no enzyme control). After incubation for 12 h, the digestion mixtures were extracted with equal volumes of ethyl acetate three times, and the organic phase was then evaporated to dryness using a vacuum evaporator (EYELA, Tokyo, Japan). The re-dissolved samples in methanol were 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).