Industrial Biotransformations

Naphthalene dioxygenase
Pseudomonas putida
1) Reaction conditions
medium: aqueous
reaction type: redox reaction
catalyst: whole cells
enzyme: naphthalene, NADH:oxygen oxidoreductase (naphthalene 1,2-dioxygenase,
naphthalene oxygenase)
strain: Pseudomonas putida
CAS (enzyme): [9014–51–1]
2) Remarks
1 = 1H-indole
2 = 2,3-dihydro-1H-indole-2,3-diol
Fig. 1.13.11.11 – 1
N
H
E
+H2O
+ 1 /2 O2
1 2
EC 1.13.11.11
● This process demonstrates the potential of biological synthesis by constructing an adequate
host cell.
● Several problems have to be solved for an effective synthesis of indigo (see figure: 1.13.11.11 – 4:
1) The activity of naphthalene dioxygenase is very low in Pseudomonas putida and even lower
in the first recombinant E. coli strains.
2) The half-life of the indole oxidizing system is only about one to two hours. Indole concentrations
above 400 mg · L –1 are toxic to cells due to inactivation of the ferredoxin component
of the enzyme system.
3) Indole is too expensive for commercial application. Alternatively tryptophan can be used
with a possible tryptophanase step (EC 4.1.99.1; [9024-00–4]) for synthesizing the indole in
the cell. But tryprophan is also too expensive.
● To overcome all these problems several improvements were necessary:
1) The dioxygenase system is improved by using better plasmids with stronger promoters.
2) The cloning of the ferredoxin producing genes results firstly not only in a more stable ferredoxin
system but in a higher concentration of ferredoxin units. Secondly site-directed mutagenesis
leads to a more stable ferredoxin system.
3) The tryptophanase system is improved by site-directed mutagenesis.
● All improvements are combined in one single host to produce indigo from glucose. Further
improvements of basic fermentation parameters lead to higher indole concentrations.
N
H
OH
OH
Genencor International, Inc.
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