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Rational Protein Design of Paenibacillus barcinonensis Esterase EstA for Kinetic Resolution of Tertiary Alcohols

Authors

  • Arnau Bassegoda,

    1. Institute of Biochemistry, Department of Biotechnology & Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Str. 4, 17487 Greifswald (Germany), Fax: (+49) 3834-86-80066
    2. Department of Microbiology, University of Barcelona, Av. Diagonal 645, 08028 Barcelona (Spain)
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  • Giang-Son Nguyen,

    1. Institute of Biochemistry, Department of Biotechnology & Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Str. 4, 17487 Greifswald (Germany), Fax: (+49) 3834-86-80066
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  • Marlen Schmidt Dr.,

    1. Institute of Biochemistry, Department of Biotechnology & Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Str. 4, 17487 Greifswald (Germany), Fax: (+49) 3834-86-80066
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  • Robert Kourist Dr.,

    1. Institute of Biochemistry, Department of Biotechnology & Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Str. 4, 17487 Greifswald (Germany), Fax: (+49) 3834-86-80066
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  • Pilar Diaz Prof. Dr.,

    1. Department of Microbiology, University of Barcelona, Av. Diagonal 645, 08028 Barcelona (Spain)
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  • Uwe T. Bornscheuer Prof. Dr.

    1. Institute of Biochemistry, Department of Biotechnology & Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Str. 4, 17487 Greifswald (Germany), Fax: (+49) 3834-86-80066
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Abstract

Protein engineering is a very powerful tool to optimize enzymes for specific applications and thus provide important chiral building blocks such as tertiary alcohols. By use of structural comparisons, esterase from Paenibacillus barcinonensis (EstA) was engineered to convert tertiary alcohol esters with excellent enantioselectivity. Whereas the wild-type enzyme converts 1,1,1-trifluoro-2-phenylbut-3-yn-2-yl acetate with very low activity and enantioselectivity (E=12, at 4 °C), several mutants show a significantly increased enantioselectivity, for example E>100 for mutant EstA–AGA, under the same reaction conditions. Furthermore, the range of tertiary alcohols obtained in enantiopure form was also broadened for EstA mutants.

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