Functional Expression and Characterization of a Panel of Cobalt and Iron-Dependent Nitrile Hydratases

Abstract

Nitrile hydratases (NHase) catalyze the hydration of nitriles to the corresponding amides. We report on the heterologous expression of various nitrile hydratases. Some of these enzymes have been investigated by others and us before, but sixteen target proteins represent novel sequences. Of 21 target sequences, 4 iron and 16 cobalt containing proteins were functionally expressed from Escherichia coli BL21 (DE3) Gold. Cell free extracts were used for activity profiling and basic characterization of the NHases using the typical NHase substrate methacrylonitrile. Co-type NHases are more tolerant to high pH than Fe-type NHases. A screening for activity on three structurally diverse nitriles was carried out. Two novel Co-dependent NHases from Afipia broomeae and Roseobacter sp. and a new Fe-type NHase from Gordonia hydrophobica were very well expressed and hydrated methacrylonitrile, pyrazine-carbonitrile, and 3-amino-3-(p-toluoyl)propanenitrile. The Co-dependent NHases from Caballeronia jiangsuensis and Microvirga lotononidis, as well as two Fe-dependent NHases from Pseudomonades, were—in addition—able to produce the amide from cinnamonitrile. Summarizing, seven so far uncharacterized NHases are described to be promising biocatalysts.

The sustainable synthesis of levetiracetam by an enzymatic dynamic kinetic resolution and an ex-cell anodic oxidation

“…S7 †). The reason was not fast deactivation of CtNHase, 30,31 but the formation of a side product: propanal and cyanide form 2-hydroxybutanenitrilea competitive substrate for CtNHase which gives 2-hydroxybutanamide upon hydration, as confirmed by HPLC-MS (ESI Fig. S8 †).…”

“…Screening a panel of known and new NHases, 30 we identified the thermotolerant cobalt-dependent Comamonas testosteroni NHase (CtNHase) as the most promising enzyme candidate (ESI Table S1 †). 31 It showed activity from pH 6.0 to 9.5 (ESI Fig.…”

High-Level Expression of Nitrile Hydratase in Escherichia coli for 2-Amino-2,3-Dimethylbutyramide Synthesis

“…However, a complex posttranslational procedure, including ion insertion and cysteine oxidation, is needed for NHase maturation and its mechanism has not been fully understood yet [7]. Recombinant expression of NHase in E. coli was usually limited by the low expression level as well as the formation of inactive and insoluble proteins [14][15][16]. To overcome these obstacles, ribosome-binding site engineering, molecular chaperone co-expression and tag fusion strategy are employed [7].…”