Novel cyanide-hydrolyzing enzyme from Alcaligenes xylosoxidans subsp. denitrificans

Abstract

A cyanide-metabolizing bacterium, strain DF3, isolated from soil was identified as Alcaligenes xylosoxidans subsp. denitrificans. Whole cells and cell extracts of strain DF3 catalyzed hydrolysis of cyanide to formate and ammonia (HCN + 2H20-HCOOH + NH3) without forming formamide as a free intermediate. The cyanide-hydrolyzing activity was inducibly produced in cells during growth in cyanide-containing media. Cyanate (OCN-) and a wide range of aliphatic and aromatic nitriles were not hydrolyzed by intact cells of A. xylosoxidans subsp. denitrificans DF3. Strain DF3 hydrolyzed cyanide with great efficacy. Thus, by using resting induced cells at a concentration of 11.3 mg (dry weight) per ml, the cyanide concentration could be reduced from 0.97 M (approximately 25,220 ppm) to less than 77 nM (approximately 0.002 ppm) in 55 h. Enzyme purification established that cyanide hydrolysis by A. xylosoxidans subsp. denitrificans DF3 was due to a single intracellular enzyme. The soluble enzyme was purified approximately 160-fold, and the first 25 NH2-terminal amino acids were determined by automated Edman degradation. The molecular mass of the active enzyme (purity, >97% as determined by amino acid sequencing) was estimated to be >300,000 Da. The cyanide-hydrolyzing enzyme of A. xylosoxidans subsp. denitrificans DF3 was tentatively named cyanidase to distinguish it from known nitrilases (EC 3.5.5.1) which act on organic nitriles.

Degradation of tetracyanonickelate (II) by Cryptococcus humicolus MCN2

“…Another cyanide degradation pathway has been elucidated in Alcaligenes xylosooxidans sub sp. denitri¢cans [21] and P. £uorescens [22]. They have a onestep reaction (Eq.…”

The nitrilase family of CN hydrolysing enzymes - a comparative study

“…SDS‐PAGE indicated the enzyme consisted of two subunits of 39 and 40 kDa with identical NH 2 –terminal sequences. The possibility that the slightly different molecular masses are due to post‐translational modification of the protein or proteolytic degradation was not excluded in this study (Ingvorsen et al. 1991).…”

Hydrolysis of nitriles by soil bacteria: variation with soil origin

“…M195 and M208 were similar to Bacillus cereus, which is a known nitrile converter (Saroja et al 2000), while M148 was closely clustered to Alcaligenes faecalis, which is also known to convert nitriles (Yamamoto et al 1991). Species of Bacillus and Alcaligenes have been isolated from cyanide containing mine tailings previously (Ingvorsen et al 1991;Meyers et al 1991Meyers et al , 1993.…”