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Use of 'Small but Smart' Libraries to Enhance the Enantioselectivity of an Esterase From Bacillus Stearothermophilus Towards tetrahydrofuran-3-yl Acetate

Use of 'Small but Smart' Libraries to Enhance the Enantioselectivity of an Esterase From Bacillus Stearothermophilus Towards tetrahydrofuran-3-yl Acetate

Alberto Nobili, Markus G Gall, Ioannis V Pavlidis, Mark L Thompson, Marlen Schmidt, Uwe T Bornscheuer

FEBS J. 2013 Jul;280(13):3084-93.

PMID: 23331978

Abstract:

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP9016186-A	Esterase from Bacillus stearothermophilus		9016-18-6	Price

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