Increasing the substrate specificity of geobacillus stearothermophilus LDH by using iterative saturation mutagenesis

Importance of chiral α-hydroxy acids (AHAs) are steadily increasing more in particular for the production of pharmaceutical and cosmetic dermatology. AHAs are indispensable for biotechology due to their importance in the production intermediate component for pharmaceutical industry. They are the main components of cosmetic-personal care product and also they have dermatological constructive effects. Chiral compounds are produced by using enzymes as biocatalyst in industrial biotechnological processes. However, enzymes are not stable under harsh conditions of the industry and recognizing a very small number of chemicals as substrate that has been limited the kemoenzimic production of industrially important chiral AHAs as a single enantiomer. Wild type Geobacillus stearothermophillus lactate dehydrogenase (bsLDH) can catalyze only the production of lactate from reduced chiral AHA. Therefore, enzyme engineering applications are needed for bsLDH which will recognize different chiral AHAs as substrate. In the proposed project, due to the high thermal resistance of bsLDH enzymes have been used. In this study, Iterative Saturation Mutagenesis (ISM) was used in order to expand the substrate range of the enzyme. ISM, only focuses on amino acid position(s) of enzyme defined by experimental or computational studies functional or amino acid position(s) having potential of being functional. Then, ISM forms mutant libraries which is limited with mutations based on candidate amino acid position(s). Due to targeting a few number of selected functional amino acids, formed mutant libraries by ISM are numerically much smaller than obtained libraries by directed mutations techniques. By selecting the best mutant from each mutant candidate library, ISM makes combinations for target mutant enzyme. Thus, ISM was repeated until obtaining the hit target enzyme. So ISM reduces studies in the selection steps in a huge rate and makes much more possible to obtain mutants having the desired properties. In this project, we aimed to produce bsLDH for introducing different industrially important target AHA as a substrate. For this purpose six mutant libraries were designed in an effort to expand the substrate range of bsLDH. The eight variants were identified as having enhanced activity toward the selected α-keto acids (oxaloacetate, benzoylformate, phenylpyruvate), belongs to the same library. These new variants now may be useful biocatalysts for chiral synthesis of α-hydroxy acids.