Biotransformation of free cyanide to formic acid by a cyanide hydratase−formamidase cascade reaction

Abstract

Free cyanide (fCN) consisting of HCN and CN- is highly hazardous. Today, removal of cyanide from industrial (mining, plating, coke-plant) wastewaters largely relies on physicochemical processes followed by microbial degradation. Enzymatic processes are gaining ground but are still at a low technological stage. The cyanide-converting enzymes of interest are primarily cyanide dihydratases (CynDs; EC 3.5.5.1.), which hydrolyze HCN to formic acid and ammonia, and cyanide hydratases (CynHs; EC 4.2.1.66), which hydrate HCN to formamide. CynHs usually have much higher specific activities and a broader pH profile especially in the alkaline region compared to CynDs. However, the product of CynH, formamide, although much less toxic than fCN, still poses a significant health risk. Therefore, it is attractive to combine the CynH with an amidase that converts formamide to formic acid and ammonia. Here we demonstrate on a laboratory scale a two-step “one-pot” detoxification of fCN (5 mM) to formic acid using recombinantly produced purified enzymes − CynH from Exidia glandulosa and formamidase (EC 3.5.1.49) from Bacillus cereus. The reaction proceeds at pH 9.0–10.0, which reduces the risk of HCN escape. We also hypothesize that the cascade can be used for fCN determination after coupling an NAD-dependent formate dehydrogenase.