N-(Benzyloxycarbonyl)-l-aspartyl-l-phenylalanine methyl ester production by immobilized Nocardia opaca: Thermolysin under hydrogen high pressure in a water-organic solvent system

N-(Benzyloxycarbonyl)-l-aspartyl-l-phenylalanine methyl ester (Z-APM) has been produced from phenylpyruvate methyl ester (PPyOMe) and N-(benzyloxycarbonyl)-aspartic acid (Z-Asp) under hydrogen high pressure in a water-ethyl acetate system. Z-APM was produced in a two step reaction using immobilized Nocardia opaca and thermolysin. Phenylalanine methyl ester (PheOMe) was produced from PPyOMe, NH₄Cl and H₂ by immobilized N. opaca. The PheOMe production rate increased linearly with increasing hydrogen pressure. The PheOMe production rate was 0.41 μmol/min/g dry cells under 100 atm of hydrogen. Z-APM was produced from PheOMe and Z-Asp by immobilized thermolysin. The highest activity (22.2 μmol/min/g thermolysin) was obtained by coupling N. opaca immobilized in calcium alginate and thermolysin immobilized in photo-crosslinkable resin prepolymer ENTP-4000. When repeated use was carried out without reactivation of the immobilized N. opaca, Z-APM production was not observed after triple use. Therefore, immobilized N. opaca was separated from immobilized thermolysin and reactivated periodically in nutrient broth for 20 h. As a result, the immobilized system retained 72% of the initial activity after being used 5 times for over 100 h.