Industrial Biotransformations

Amidase
Klebsiella oxytoca
F 3C
OH
1) Reaction conditions
[1]: 0.636 M, 100 g · L –1 [157.09 g · mol –1 ]
pH: 8.0
T: 37 °C
reaction type: carboxylic acid amide hydrolysis
catalyst: suspended whole cells
enzyme: acylamide amidohydrolase (amidase)
strain: E. coli strain containing recombinant Klebsiella oxytoca amidase
CAS (enzyme): [9012 ± 56-0]
2) Remarks
CONH 2
1
● The amidase was purified and characterized from wild-type strain Klebsiella oxytoca PRS1.
● The amidase gene was cloned into E. coli to improve safety and the productivity of the biotransformation.
In addition, this avoids the slime-capsule problem and gives the possibility to use
other microorganisms as hosts for the cloned gene.
3) Flow scheme
Not published.
E1
2
EC 3.5.1.4
1 = 2-(trifluoromethyl)-2-hydroxypropanamide
2 = (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropanoic acid
3 = (S)-2-(trifluoromethyl)-2-hydroxypropanamide Lonza AG
Fig. 3.5.1.4 – 1
HOOC
OH
CF 3
4) Process parameters
yield: almost the theoretical maximum of 50 %
ee: 100 %
optical purity > 98 % of (R)-acid
reactor volume: 1500 L
capacity: (R)-acid: 100 kg; (S)-acid: 100 g
down stream processing: Microfiltration to remove cells. Ultrafiltration through a 70-kDa membrane
to remove proteins. Concentration by thin-film evaporation.
Extraction of (S)-amide with ethyl acetate. Extraction of (R)-acid with
methyl tert-butyl ether. Vacuum distillation of ether. The precipitated
product was collected by centrifugation and dried at 30 °C and
p 50 mbar. The drying conditions must be closely controlled to avoid
sublimation of the product.
382
+
F 3C
OH
3
CONH 2