Industrial Biotransformations

Phenylalanine dehydrogenase / Formate dehydrogenase
Thermoactinomyces intermedius / Candida boidinii
1) Reaction conditions
[1]: 0.266 M, 50 g · L –1 [188.18 g · mol –1 ]
[2]: 19.49 g · L –1 ammonium formate
[3]: 0.35 g · L –1 NAD
pH: 8.0
T: 40 °C
medium: aqueous
reaction type: reductive amination
catalyst: heat-dried cells of Escherichia coli containing cloned PheDH from Thermoactinomyces
intermedius and heat-dried cells of Candida boidinii, or dried recombinant
Pichia pastoris containing Thermoactinomyces intermedius PheDH
enzyme: l-phenylalanine dehydrogenase
enzyme: formate dehydrogenase
strain: Thermoactinomyces intermedius / Candida boidinii / Pichia pastoris
CAS (enzyme): [69403-12-9] PheDH / [9028-85-7] FDH
2) Remarks
O E1
O
1 2
1 = 5-(1,3-dioxolan-2-yl)-2-oxopentanoic acid
2 = (S)-2-amino-5-(1,3-dioxolan-2-yl)pentanoic acid
Fig. 1.4.1.20 / 1.2.1.2 – 1
O
O
COOH
CO 2
NADH NAD +
● FDH activity in P. pastoris was 2.7-fold greater than for C. boidinii.
E2
HCO 2NH 4
EC 1.4.1.20 / 1.2.1.2
Bristol-Myers Squibb
● Phenylalanine dehydrogenase was cloned and overexpressed in Escherichia coli and Pichia pastoris.
● Fermentation of T. intermedius yielded 184 units of PheDH activity per liter of whole broth in
6 h. In contrast, E. coli BL(DE3)pPDH155k produced over 19,000 units per liter of whole broth
in about 14 h.
● Acetal amino acid had been previously prepared by an eight-step chemical synthesis from 3,4dihydro[2H]pyran.
● An alternate synthesis was demonstrated by the reductive amination of ketoacid acetal using
purified enzyme PheDH from Thermoactinomyces intermedius. The reaction required ammonia
and NADH; NAD + produced during the reaction was recycled to NADH by the oxidation of
formate to CO 2 using purified formate dehydrogenase (FDH).
H 2N
O
COOH
207