Use of 'small but smart' libraries to enhance the enantioselectivity of an esterase from Bacillus stearothermophilus towards tetrahydrofuran‐3‐yl acetate. (14th February 2013)

• Record Type: Journal Article
• Authors: Nobili, Alberto; Gall, Markus G.; Pavlidis, Ioannis V.; Thompson, Mark L.; Schmidt, Marlen; Bornscheuer, Uwe T.
• Is Part Of: FEBS journal. Volume 280:Number 13(2013)
• Page Start: 3084
• Page End: 3093
• Publication Date: 2013-02-14
• Subjects: Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.12137
• Languages: English
• ISSNs: 1742-464X

Abstract

<abstract abstract-type="main" id="febs12137-abs-0001"> <title> <x xml:space="preserve">Abstract</x> </title> <p>Two libraries of simultaneous double mutations in the active site region of an esterase from <italic>Bacillus stearothermophilus</italic> 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 3<sc>dm</sc>. 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 (<italic>E</italic> = 4.3) in the hydrolysis of (<italic>S</italic>)‐tetrahydrofuran‐3‐yl acetate, the best variants obtained with this approach showed increased <italic>E</italic>‐values of up to 10.4. Furthermore, some variants with inverted enantiopreference were found.</p> </abstract>