THWRC Awarded Proposal 513TAM0032H

Project number:           513TAM0032H

Title:                             Cyanide Remediation: Evolving Improved Enzymes

Lead PI:                         Michael Benedik

Awarded amount:        $40,000


Project Abstract

Cyanide is used extensively in a variety of industrial applications. As the simplest nitrile it has many uses in the production of plastics and numerous nitrile derived pharmaceuticals. More importantly though, by virtue of its ability to form tight complexes with a variety of metals, cyanide has been extensively used in mining for the extraction of metals from ore, especially gold. This ability to form complexes with metal and thereby keep them in solution makes the use of cyanide invaluable in the metal plating industry.

            These applications in mining and plating lead to the cyanide contamination of both environmental sites as well as production of cyanide contaminated water. As cyanide is a potent respiratory poison, these large quantities of waste water need to be treated before their release. The long term goal of our work is to develop a cost-competitive, effective, and simple method for the remediation of cyanide contaminated waste-water. The basis for our work is the utilization of microbial enzymes that have the capacity to degrade cyanide, in other words a bioremediation solution.

            The enzymes we use are microbial nitrilases which are a family of enzymes that convert nitriles to other compounds generally through a hydration reaction. Cyanide being the simplest of the nitriles, is converted to either formamide or formate and ammonia depending upon the enzyme used. These enzymes are well suited for remediation applications in that (1) they are stable over long time periods (2) require no co-factors for their operation (3) and function well as purified enzyme, crude extracts, or within living or dead cells and (4) are readily expressed at high levels in microbial systems. My lab has successfully cloned and expressed 6 different members of this enzyme family, each with slightly different properties. The enzymes are stable, retaining full activity over at week at 37C. We have shown these enzymes are capable of eliminating all detectable cyanide from heavily contaminated water from silver plating baths, but required the reduction of pH to below 8.5.

Recently our research has focused on using high throughput mutagenesis methods to generate mutants of these nitrilases that extend the operational pH range similar to conditions found in the field. Generally cyanide solutions are kept at high pH to reduce the volatilization of cyanide to HCN gas which occurs below pH 9. We have reported (Wang et al, 2012) on the isolation and characterization of such mutants in the B. pumilus enzyme and learned from these mutants is that stability of the oligomeric form (quaternary structure of the enzyme) is the limiting factor in stability and pH tolerance.

            A cluster of stabilizing mutants reside in the c-terminal portion of the enzyme. We hypothesize that this domain is crucial for locking the oligomer into a tight configuration, enhancing stability. The construction of a hybrid protein replacing the c-terminal domain with that from a different species led to a protein of dramatically enhanced stability, much more significant than any mutant isolated to date. The work in this proposal aims to (1) continue the complete characterization of that hybrid mutant; (2) combine other stabilizing mutants into that hybrid to determine whether any are synergistic; (3) continue our studies on the oligomerizing region of the protein.