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Expression and stabilization of galactose oxidase in Escherichia coli by directed evolution

2001; Oxford University Press; Volume: 14; Issue: 9 Linguagem: Inglês

10.1093/protein/14.9.699

1741-0134

Lianhong Sun, Ioanna P. Petrounia, Makoto Yagasaki, Geethani Bandara, Frances H. Arnold,

Enzyme Catalysis and Immobilization

We have used directed evolution methods to express a fungal enzyme, galactose oxidase (GOase), in functional form in Escherichia coli. The evolved enzymes retain the activity and substrate specificity of the native fungal oxidase, but are more thermostable, are expressed at a much higher level (up to 10.8 mg/l of purified GOase), and have reduced negative charge compared to wild type, all properties which are expected to facilitate applications and further evolution of the enzyme. Spectroscopic characterization of the recombinant enzymes reveals a tyrosyl radical of comparable stability to the native GOase from Fusarium.