Regiospecific C–H amination of (−)-limonene into (−)-perillamine by multi-enzymatic cascade reactions

Bioresources and Bioprocessing

Table 1 Optimization of reaction conditions of Module 1

Entry	Temp. (°C)	pH	Sub. conc. (mM)	Cell dosage (gcdw/L)	(−)-Perillyl alcohol (mM)
1	30	8.0	5.0	10	1.0
2	30	7.5	5.0	10	1.4
3	30	8.5	5.0	10	0.9
4	25	7.5	5.0	10	2.7
5	25	7.5	5.0	5	2.8
6	25	7.5	5.0	15	2.0
7	25	7.5	5.0	20	1.9
8^a	25	7.5	5.0	5	1.9
9^a	25	7.5	5.0	10	2.9
10^a	25	7.5	5.0	15	4.6
11^a	25	7.5	5.0	20	2.7
12^a	25	7.5	7.5	15	5.1
13^a	25	7.5	10	15	6.8
14^a	25	7.5	15	15	6.7

Reaction conditions (0.5 mL): ( −)-limonene (5–10 mM), A7F resting cells (5–20 g_cdw/L), sodium formate (100 mM), DMSO (2% v/v), KPB buffer (pH 7.5, 100 mM) or Tris–HCl buffer (pH 8.0–8.5, 100 mM). The reaction mixtures were shaken at 25 or 30 °C and 800 rpm for 4 h
^aAdditional 1 mg/mL of Triton X-100 was added. Temp.: temperature; Sub. conc.: substrate concentration