Use of ‘small but smart’ libraries to enhance the enantioselectivity of an esterase from Bacillus stearothermophilus towards tetrahydrofuran‐3‐yl acetate

• Published in: The FEBS journal 2013-07, Vol.280 (13), p.3084-3093
• Main Authors: Nobili, Alberto, Gall, Markus G., Pavlidis, Ioannis V., Thompson, Mark L., Schmidt, Marlen, Bornscheuer, Uwe T.
• Format: Article
• Language: English
• Subjects: Acetates - chemistry; Acetates - metabolism; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Carboxylesterase - chemistry; Carboxylesterase - genetics; Carboxylesterase - metabolism; Catalytic Domain; Computational Biology - methods; Databases, Protein; directed evolution; Directed Molecular Evolution; enantioselectivity; esterase; Furans - chemistry; Furans - metabolism; Gene Library; Geobacillus stearothermophilus - enzymology; Hydrology; Hydrolysis; Kinetics; Models, Molecular; Molecular Conformation; Molecular Docking Simulation; Mutant Proteins - chemistry; Mutant Proteins - metabolism; Mutation; Protein Engineering - methods; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; secondary alcohol; Stereoisomerism; Substrate Specificity; tetrahydrofuran‐3‐yl acetate
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.12137

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. A semi‐rational approach was applied for the enhancement of the enantioselectivity of an esterase from Bacillus stearothermophilus towards the industrially interesting substrate tetrahydrofuran‐3‐yl acetate, based on data derived from structural alignment. The design of ‘small but smart’ libraries led to a 2.4‐fold increase of (S)‐selectivity compared to wild type enzyme, while some mutants with marginal (R)‐selectivity were found.