The key advantage of using VTU´s Pichia system for the generation of enzyme variants is that engineering is performed in the final production host. Therefore, potential bottlenecks in changing the host system from primary screen to final enzyme production are eliminated, thus accelerating the commercialization of your improved enzyme. In addition, VTU´s established high-throughput microscale cultivation and screening protocols facilitate the rapid identification of improved protein variants with specific properties. Application of VTU´s Pichia toolbox ensures production of high-value enzymes at lower manufacturing costs.
Next to classical objectives like increasing activity towards a specific substrate, any kind of increased stability upon incubation at e.g. varying temperatures, differing pH-values or diverse reaction matrices has been successfully achieved.
Libraries of gene variants are created by error-prone PCR and/or site directed mutagenesis potentially supported by molecular modeling. The formation of single copy integrations is favored by using appropriate amounts of linearized DNA paired with the ideal selection pressure in order to allow for highest comparability in screenings. Using VTU´s established 96-deep-well plate cultivation for homogeneous growth of individual transformants, high-throughput screening of thousands of gene variants in parallel is carried out using spectrophotometric or fluorometric activity analysis. In addition, a pre-screening on solid media to eventually cultivate only a selected set of transformants can be envisaged.