GENETIC OPTIMIZATION FOR ALKALINE pH OF A CYANIDE DIHYDRATASE FROM PSEUDOMONAS STUTZERI

Abstract

Even though cyanide is highly toxic, it is commonly found in industrial waste generated by several industries such as metal plating and mining. This chemical is hazardous for many organisms, including mammals, because it inhibits key factors in the respiratory pathway. Surprisingly, several microorganisms can degrade and even survive cyanide’s presence. These microbes, fungi and bacteria, count on a variety of cyanide degrading enzymes that break cyanide into less toxic compounds. An example is the cyanide dihydratase (CynDstut) found in Pseudomonas stutzeri AK61. The enzyme operates optimally around pH 7-8, but most polluted waters have much higher alkaline pH. The aim of this research project is to construct genetic mutants for this enzyme that are able to operate in these highly alkaline environments. The DNA that encodes the enzyme has been cloned into common Escherichia coli where it makes functional protein. We show how using methods such as error prone PCR amplification that creates mutations, strains of E. coli were screened for cyanide degrading activity at pH 10 and how we identified a mutant that tolerates these conditions. These novel mutant enzymes can then be analyzed for improved properties useful for bioremediation of cyanide waste waters.