Expression and stabilization of galactose oxidase in Escherichia coli by directed evolution.
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.
Sun
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Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125, USA.
Petrounia
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Yagasaki
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Bandara
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Arnold
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D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D019020:Directed Molecular Evolution / Q000379:methods*; D004563:Electrochemistry; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005670:Fusarium / Q000201:enzymology; D005692:Galactose Oxidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D014644:Genetic Variation; D006358:Hot Temperature; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D014169:Transformation, Bacterial
D011994:Recombinant Proteins; D005692:Galactose Oxidase