Exploring the Potential of Fungal Arylacetonitrilases in Mandelic Acid Synthesis

Abstract

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 (<1% of the total product). This enzyme produced up to >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.

Process Development for the Production of (R)-(−)-Mandelic Acid by Recombinant Escherichia coli Cells Harboring Nitrilase from Burkholderia cenocepacia J2315

“…Compared with bacterial nitrilases, fungal nitrilases performed better in the hydrolysis of high concentrations of mandelonitrile. High conversions (75–93%) were achieved when hydrolyzing 500 mM of mandelonitrile with three fungal nitrilases, including NitAn, NitAb, and NitAc . The optical purity of mandelic aicd correlated inversely with the mandelonitrile concentration for nitrilase BCJ2315.…”

“…Veselá et al compared the yield (%), product concentration (g/L), volume productivity (g/L·d), and ee value (%) of the product and catalyst productivity (g/g·dcw) of nitrilase from different sources in various biocatalytic process types (batch, fed-batch, and biphasic system) . The potential of nitrilases from Alcaligenes sp., , A. faecalis , B. cenocepacia J2315, and Aspergillus niger as biocatalysts in the form of recombinant whole cells or immobilized recombinant cells were compared in detail.…”

“…High conversions (75−93%) were achieved when hydrolyzing 500 mM of mandelonitrile with three fungal nitrilases, including NitAn, NitAb, and NitAc. 19 The optical purity of mandelic aicd correlated inversely with the mandelonitrile concentration for nitrilase BCJ2315. The ee value decreased with increasing substrate concentration.…”

Constitutive secretory expression and characterization of nitrilase from Alcaligenes faecalis in Pichia pastoris for production of R‐mandelic acid

“…Consequently, it has become possible to conduct in-silico mining of new efficient nitrilases from a large number of database resources, which gradually replaced the traditional nitrilase screening strategies. The discovery and screening, 2,[5][6][7] cloning and expression, 8,9 and molecular modification [10][11][12][13] of numerous nitrilases has been reported. In recent years, the expression of nitrilases in recombinant Escherichia coli has increasingly relied on constitutive promoters to avoid the need for adding expensive chemical inducers.…”