Expression and stabilization of galactose oxidase in Escherichia coli by directed evolution |
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Authors: | Sun Lianhong; Petrounia Ioanna P; Yagasaki Makoto; Bandara Geethani; Arnold Frances H |
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Affiliation: | Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125, USA |
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Abstract: | We have used directed evolution methods to express a fungalenzyme, galactose oxidase (GOase), in functional form in Escherichiacoli. The evolved enzymes retain the activity and substratespecificity of the native fungal oxidase, but are more thermostable,are expressed at a much higher level (up to 10.8 mg/l of purifiedGOase), and have reduced negative charge compared to wild type,all properties which are expected to facilitate applicationsand further evolution of the enzyme. Spectroscopic characterizationof the recombinant enzymes reveals a tyrosyl radical of comparablestability to the native GOase from Fusarium. |
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