Semi-rational design of geobacillus stearothermophilus l-lactate dehydrogenase to access various chiral ?-hydroxy acids

Binay, Barış (Arel Author)Chiral ?-hydroxy acids (AHAs) are rapidly becoming important synthetic building blocks, in particular for the production of pharmaceuticals and other fine chemicals. Chiral compounds of a variety of functionalities are now often derived using enzymes, and L-lactate dehydrogenase from the thermophilic organism Geobacillus stearothermophilus (bsLDH) has the potential to be employed for the industrial synthesis of chiral ?-hydroxy acids. Despite the thorough characterization of this enzyme, generation of variants with high activity on nonnatural substrates has remained difficult and therefore limits the use of bsLDH in industry. Here, we present the engineering of bsLDH using semi-rational design as a method of focusing screening in a small and smart library for novel biocatalysts. In this study, six mutant libraries were designed in an effort to expand the substrate range of bsLDH. The eight variants identified as having enhanced activity toward the selected ?-keto acids belonged to the same library, which targeted two positions simultaneously. These new variants now may be useful biocatalysts for chiral synthesis of ?-hydroxy acids