Cyanide hydratase from Aspergillus niger K10: Overproduction in Escherichia coli, purification, characterization and use in continuous cyanide degradation
Abstrakt
A cyanide hydratase from Aspergillus niger K10 was expressed in Escherichia coli and purified. Apart from HCN, it transformed some nitriles, preferentially 2-cyanopyridine and fumaronitrile.
V-max and K-m for HCN were ca. 6.8mmol min(-1) mg(-1) protein and 109mM, respectively. V-max for fumaronitrite and 2-cyanopyridine was two to three orders of magnitude lower than for HCN (ca. 18.8 and 10.3 mu.,mol min(-1) mg(-1), respectively) but K-m was also lower (ca. 14.7 and 3.7 mM, respectively).
Both cyanide hydratase and nitrilase activities were abolished in truncated enzyme variants missing 18-34 C-terminal aa residues. The enzyme exhibited the highest activity at 45 degrees C and pH 8-9; it was unstable at over 35 degrees C and at below pH 5.5.
The operational stability of the whole-cell catalyst was examined in continuous stirred membrane reactors with 70-mL working volume. The catalyst exhibited a half-life of 5.6 h at 28 degrees C.
A reactor loaded with an excess of the catalyst was used to degrade 25 mM KCN. A conversion rate of over 80% was maintained for 3 days.