Synthesis of allysine ethylene acetal using phenylalanine dehydrogenase from Thermoactinomyces intermedius

Allysine ethylene acetal [(S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid (2)] was prepared from the corresponding keto acid by reductive amination u sing phenylalanine dehydrogenase (PDH) from Thermoactinomyces intermedius A TCC 33205. Glutamate, alanine, and leucine dehydrogenases, and PDH from Spo rosarcina species (listed in order of increasing effectiveness) also gave t he desired amino acid but were less effective. The reaction requires ammoni a and NADH. NAD produced during the reaction was recyled to NADH by the oxi dation of formats to CO2 using formate dehydrogenase (FDH). PDH was produce d by growth of T. intermedius ATCC 33205 or by growth of recombinant Escher ichia coli or Pichia pastoris expressing the Thermoactinomyces enzyme. Usin g heat-dried T. intermedius as a source of PDH and heat-dried Candida boidi nii SC13822 as a source of FDH, reaction yields averaged 84 M%, and ee was >98%, but production of T. intermedius could not be scaled up. Using heat-d ried recombinant E. coli as a source of PDH and heat-dried Candida boidinii as a source of FDH, 197 kg of 2 was produced in three batches with an aver age yield of 91 M% and ee > 98%. In a third generation process, heat-dried methanol-grown P. pastoris expressing endogenous FDH and recombinant Thermo actinomyces PDH was used to produce 15 kg of 2 with a yield of 97 M% and >9 8% ee. (C) 2000 Elsevier Science Inc. All rights reserved.