The Cyanide Metabolism Of Alcaligenes Sp. DN25, And Cloning,Expression Of Its Cyanide-degrading Enzyme

Alcaligenes sp. DN25, preserved in the lab, showed a high biodegradation activity for cyanide. The strain with the favorable capability to alkaline condition and cyanide tolerence, offers new perspectives in the bioremediation of cyanide-containing effluents.The strain of Alcaligenes sp. DN25, was able to utilize cyanide, including potassium cyanide, acetonitrile, potassium thiocyanate and potassium ferrocyanide, as the sole source of nitrogen. The degradation rate of KCN was relative to the pH, initial cyanide concentration and the bacterial inoculum size. The strain showed good degradability in alkaline media, up to an initial pH of9.7. At the optimum pH of8.0, the cyanide concentration could be reduced from43mM (equal to1,100ppm CN") to less than8.3μM in12hours. The maximum initial degradation rate (IDR) was14.58mM·h-1by the inoculum size of5%and the initial cyanide concentration of25mM. The minimal inhibitory concentration (MIC) of CN-for the strain was determined and showed a similar value to a fungus Fusarium solani IHEM8026. Cyanide was biodegraded to non-toxic endproducts, including formate, formamide and ammonia, via a hydrolytic pathway. Formamide and ammonia were proved to be further utilized by Alcaligenes sp. DN25. The gene of cyanide-degrading enzyme was cloned from chromosomal DNA of Alcaligenes sp. DN25, and the recombinant enzyme was expressed at high levels in Escherichia coli. The electrophoresis pattern of SDS-PAGE of recombinant protein analyzed via the gel imaging analysis system suggested that the recombinant cyanide-degrading enzyme was given a calculated molecular mass of appropriate38kDa. The optimum induced expression conditions for the recombinant enzyme,28℃, IPTG concentration of0.8mM, induced time of12hours, and initial cell density of0.6at600nm, was obtained via several optimization work.Purification of the recombinant enzyme was facilitated by the addition of a N-terminal six-histidine affinity purification tag and95percent of purity was obtained via an one-step method. Cyanide was completely transformed into formate, formamide and ammonia by the the purified recombinant cyanide-degrading enzyme. The further assays of formamide hydrolysis proved that the generated formate does not come from formamide as a result of secondary hydrolysis. The results suggested that, the cyanide-degrading enzyme from Alcaligenes sp. DN25, which degraded cyanide to formate, ammonia and formamide simultaneously, maybe catalyze the cyanide degradation via two different catalytic pathways, and this situation is first reported in the current study.