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.
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