Abstrakt
The application of arylacetonitrilases from filamentous fungi to the hydrolysis of high concentrations of (R, S)-mandelonitrile (100-500 mM) was demonstrated for the first time. Escherichia coli strains expressing the corresponding genes were used as whole-cell catalysts.
Nitrilases from Aspergillus niger, Neurospora crassa, Nectria haematococca, and Arthroderma benhamiae (enzymes NitAn, NitNc, NitNh, and NitAb, respectively) exhibited different degrees of enantio-and chemoselectivity (amide formation). Their enantio-and chemoselectivity was increased by increasing pH (from 8 to 9-10) and adding 4-10 % (v/v) toluene as the cosolvent.
NitAn and NitNc were able to convert an up to 500 mM substrate in batch mode. NitAn formed a very low amount of the by-product, amide (70 g/L of (R)-mandelic acid (e.e. 94.5-95.6 %) in batch or fed-batch mode.
Its volumetric productivities were the highest in batch mode [571 +/- 32 g/(L d)] and its catalyst productivities in fed-batch mode (39.9 +/- 2.5 g/g of dcw). NitAb hydrolyzed both enantiomers of 100 mM (R, S)-mandelonitrile at pH 5.0 and is therefore promising for the enantioretentive transformation of (S)-mandelonitrile.
Sequence analysis suggested that fungal arylacetonitrilases with similar properties (enantioselectivity, chemoselectivity) were clustered together.