Industrial Biotransformations

Nitrile hydratase
Rhodococcus rhodochrous
1 = nitrile
2 = amide
3 = acid
Fig. 4.2.1.84 – 2
R
R
1
1
CN
CN
nitrile hydratase R NH2 amidase
R OH
+ H2O O
+H2O
- NH3 O
nitrilase
+ H2O
● Microorganisms that produce amidases beside the nitrile hydratase are not suitable for the
production of acrylamide without adding an amidase inhibitor.
● In the course of improvement of the biocatalyst for the production of acrylamide three main
strains were used:
1) Rhodococcus sp. N774
2) Pseudomonas chlororaphis
3) Rhodococcus rhodochrous
● The Rhodococcus sp. N774 strain was used for three years before the better Pseudomonas chlororaphis
strain was found.
● The Pseudomonas strain cannot grow on acrylonitrile but grows on isobutyronitrile.
R
2
O
3
OH
+ NH 3
EC 4.2.1.84
● The optimization of the Pseudomonas strain reveals that methacrylamide causes the greatest
induction of nitrile hydratase. The addition of ferrous or ferric ions to the culture medium
increases enzyme formation, no other ionic addition shows improvements, indicating that the
nitrile hydratase contain Fe 2+ ions as a cofactor.
● The growth medium can be optimized resulting in an amount of nitrile hydratase of 40 % of
the total soluble protein formed in the cells.
● A problem during growth of Pseudomonas chlororaphis strain in the first optimized sucrose containing
medium is the production of mucilaginous polysaccharides. These causes a high viscosity,
resulting in difficulties during cell harvest.
● Using chemical mutagenesis methods (N-methyl-N′-nitro-N-nitrosoguanidine = MNNG)
mucilage polysaccharide-non-producing mutants could be isolated. The following table shows
the improvements (total activity increases 3,000-times) by optimizing the fermentation medium
and by mutagenesis:
482
3