Cyanide is both a useful and dangerous chemical compound that serves as a crucial component in multiple industrial processes, including metal mining. The leaching process that utilizes cyanide ions to help separate target metals and increase mining yield is an industrial standard for chemical leaching. However, this method of ore extraction results in toxic cyanide waste that requires dangerous, costly, and potentially environmentally damaging remediation systems to degrade. As cyanide is a naturally occurring substance, several organisms contain enzymes capable of oxidizing cyanide into less toxic compounds. Despite the effectiveness of these proteins, they lack stability and functionality at the alkaline pH levels industrial cyanide is stored at. This project attempts to optimize the screening and mutagenesis methods in hopes of a isolating an alkaline tolerant mutant of cyanide hydratase, an enzyme originally found in the fungus Gloeocercospora sorghi. This approach incorporates random mutagenesis of the target fungal gene using error-prone polymerase chain reaction and an in vivo picric acid assay that tests the activity of the mutant enzymes at target conditions. Experimentation was used to determine the ideal conditions for a screening method by testing the activity of the wild-type positive control at different reaction conditions. The final, optimized screening conditions for the high throughput assay combined a 50 μL aliquot of cell culture grown overnight in a 96 well plate with a 50 μL of 0.1 M CAPS buffered to pH 10.5. As screening continues, these conditions can be used to identify a viable, alkaline tolerant mutant. If such a mutant is identified, the molecule would be a strong bioremediation candidate for the metal mining industry and could lead to more efficient and environmentally friendly degradation of cyanide waste
