Directed Evolution of Galactose Oxidase

Galactose oxidase (EC 1.1.3.9, GAO) from the fungus Fusarium sp. was used as a starting gene for directed evolution. The GAO has a molecular weight of 68 kDa, and is a monomeric glycoprotein with 1.7% carbohydrate, formed by 639 amino acid residues. It catalyzes the following reaction  

In the first step, the GAO gene was isolated from Fusarium sp., and fused to a strong promoter (lacZ). When the promoter is induced, transcription by RNA polymerase begins, and both mRNA and GAO are produced. 

Selected improved variants were later recombined using a method similar to DNA shuffling. Three or four rounds of mutation allowed the selection of a mutant enzyme with 15-fold higher activity, and 5°C increase in stability. Substrate selectivity was unchanged with respect to the wild-type enzyme from FusariumP This example is one of the many possible applications of directed evolution for the generation of new bioassay reagents with new or improved characteristics. 

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