Synthesis
Synthesis of 2-methyl-DL-serine hydrate: strain JM109/pSFNRHMT-Y339S was incubated overnight in LB agar medium containing 100 mg/l ampicillin and then inoculated into 50 ml of LB medium containing 100 mg/l ampicillin in 500 ml Sakaguchi flasks and incubated at 37C for 120 reciprocal/ and incubated for 17 hours. The obtained cells were collected by centrifugation (8,000 g, 10 min) and then washed with 30 ml of 50 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM pyridoxal phosphate. Then, cell suspensions were prepared using 50 ml of the same buffer. Cells collected from 2 ml of cell suspension by centrifugation (18,000 g, 10 min) were added to 10 mM formaldehyde, 30 mM ML-alanine, 0.1 mM pyridoxal phosphate, and 50 mM potassium phosphate buffer (to which 100 l of a solution consisting of pH 7.4 was added, and the reaction was carried out for 1 hr at 30C. At the end of the reaction, the supernatant obtained by centrifugation (18,000 g, 5 min, 4 C) was analyzed by ESI-MS and the molecular ion peak of 2-methyl-DL-serine hydrate was observed.
