Method of Thermostabilization of a Protein and/or Stabilization Towards Organic Solvents
Damborsky, J., Prokop, Z., Koudelakova, T., Stepankova, V., Chaloupkova, R., Gora, A., Chovancova, E., Brezovsky, J.
Masaryk University, Žerotínovo nám. 9, 601 77 Brno, Czech Republic
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The present invention relates to a method of thermostabilization of a protein and/or stabilization of a protein towards organic solvents wherein the protein contains access routes, wherein at least one amino acid in the bottleneck of the access route of the protein is mutated. Preferably, the method comprises the steps of identification of amino acids forming the bottleneck of the access routes, wherein said amino acids control the exchange of the solvent between the buried protein core and the surrounding environment and/or in the packing of the amino acids inside the access route; modification of the amino acids determined in the previous step so that the packing of the amino acids inside the tunnel is improved and the access route prevents the access of the undesired solvent molecules to the protein core, while it allows the passage of the compounds necessary at the protein core to enable the protein to perform its biological function. Preferably, the method further includes the step of assessment of the result of the modification, wherein the enzyme is analyzed in the form of whole cells containing the expressed protein, crude extract, or partially purified or purified protein.