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FEBS J. 2013 Jul;280(13):3084-93. doi: 10.1111/febs.12137. Epub 2013 Feb 14.

Use of 'small but smart' libraries to enhance the enantioselectivity of an esterase from Bacillus stearothermophilus towards tetrahydrofuran-3-yl acetate.

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Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Greifswald, Germany.


Two libraries of simultaneous double mutations in the active site region of an esterase from Bacillus stearothermophilus were constructed to improve the enantioselectivity in the hydrolysis of tetrahydrofuran-3-yl acetate. As screening of large mutant libraries is hampered by the necessity for GC/MS analysis, mutant libraries were designed according to a 'small but smart' concept. The design of focused libraries was based on data derived from a structural alignment of 3317 amino acid sequences of α/β-hydrolase fold enzymes with the bioinformatic tool 3DM. In this way, the number of mutants to be screened was substantially reduced as compared with a standard site-saturation mutagenesis approach. Whereas the wild-type esterase showed only poor enantioselectivity (E = 4.3) in the hydrolysis of (S)-tetrahydrofuran-3-yl acetate, the best variants obtained with this approach showed increased E-values of up to 10.4. Furthermore, some variants with inverted enantiopreference were found.

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