title: Use of 'small but smart' libraries to enhance the enantioselectivity of an esterase from Bacillus stearothermophilus towards tetrahydrofuran-3-yl acetate.
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.
authors: Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Greifswald, Germany.
authors: Alberto
authors: A
authors: Nobili
authors: Markus G
authors: MG
authors: Gall
authors: Ioannis V
authors: IV
authors: Pavlidis
authors: Mark L
authors: ML
authors: Thompson
authors: Marlen
authors: M
authors: Schmidt
authors: Uwe T
authors: UT
authors: Bornscheuer
mesh_terms: D000085:Acetates / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043182:Carboxylesterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D019295:Computational Biology / Q000379:methods; D030562:Databases, Protein; D019020:Directed Molecular Evolution; D005663:Furans / Q000737:chemistry / Q000378:metabolism*; D015723:Gene Library; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D062105:Molecular Docking Simulation; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D000085:Acetates; D001426:Bacterial Proteins; D005663:Furans; D050505:Mutant Proteins; D011994:Recombinant Proteins; D043182:Carboxylesterase; C483833:carboxylesterase Est30, Bacillus