Expression and characterization of a computationally designed laccase-like enzyme

Abstract : Protein engineering of oxido-reductase enzymes, such as laccases, has attracted considerable interest due to their present and potential use in industrial applications. In an effort to improve the stability of laccases for various applications, computational methods were used to design a laccase-like enzyme with optimized folding characteristics and improved stability. The laccase glycoprotein from the fungus Trametes versicolor (TvL) was used as a basis for designing a new laccase-like protein. The backbone structure of TvL was left intact while all the amino acids except for the ones around the trinuclear copper center were optimized for stability by computational methods. This newly designed laccase-like ~60 kDa amino acid chain was optimized for E. coli expression and was inserted in a pET vector with a C-terminal poly-His tag. Since this enzyme is expressed in bacteria, it will be non-glycosylated. This laccase-like enzyme has been successfully expressed in the presence of copper following IPTG induction in E. coli. The majority of the expressed protein was found in the inclusion body fraction. In order to refold the protein, different strategies of denaturation and folding have been utilized. Under the right conditions, a small amount of the protein can be found in the soluble fraction and it has been purified using metal affinity, ion exchange and gel filtration chromatography methods. The characterization of the final protein product includes molecular weight spectra (MALDI-TOF), conformational analysis (CD), thermal stability (CD) and copper content. Lastly, the kinetic activity of the enzyme under several conditions and with various substrates will be determined and compared to the original TvL laccase as well as to previously studied laccases from other sources.
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Conference papers
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Submitted on : Friday, January 11, 2013 - 12:02:03 PM
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D. J. Glykys, G. Szilvay, Pablo Tortosa, Maria Suarez, Alfonso Jaramillo. Expression and characterization of a computationally designed laccase-like enzyme. Food, Pharmaceutical & Bioengineering Division, Nov 2008, United States. pp.618c. ⟨hal-00772888⟩

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