Synthesis of kyotorphin precursor by an organic solvent-stable protease from Bacillus licheniformis RSP-09-37

The synthesis of the analgesic dipeptide kyotorphin precursor (Bz-Tyr-Arg-NH2) was studied under kinetically controlled conditions in 10–90% (v/v) aqueous-acetonitrile media at −20 °C using a novel protease obtained from the cell free supernatant of a Bacillus licheniformis mutant strain (RSP-09-37). α-Chymotrypsin (CT) was used for comparison. The conditions for maximum yield of kyotorphin precursor synthesis were optimized using CT by varying the type of nucleophile (amide and ester), nucleophile concentration and nucleophile to acyl donor ratio. The nucleophile (Arg-NH2) at a concentration 400 mM and nucleophile to acyl donor ratio 1:40 was found to be optimum for kyotorphin precursor synthesis. The protease from B. licheniformis RSP-09-37 was stable even at 90% acetonitrile concentration and allowed for a significantly higher synthesis over hydrolysis ratio (S/H ratio) of 15.6 compared to only 3.0 found for CT at −20 °C.