Gcms qp5050 system

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).

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).