Summary

The development of a screening assay by coupling respiratory activity to enzyme activity was successful and applied in library screening to validate the assay. Randomly selected cultures from were cultivated in the μTOM device. The resulting growth-coupled production can be seen as a metabolic burden in the monitored oxygen transfer rate (OTR). Furthermore, an inverse correlation was found between the growth rates of the clones (derived from the OTR) and produced protein amount. Using this correlation, clones with low production rates <0.031 g/L/h can be distinguished from clones with higher production rates >0.071 g/L/h. Model clones were cultivated in Syn6-MES medium with only phytic acid as phosphorus source. A correlation was found between the growth rates of the clones (derived from the OTR) and the offline measured phytase activity. This correlation was verified with a larger mutant library of 96 clones which were selected libraries generated by CG Schwaneberg (SSM and epPCR mutant libraries from E.coli AppaA, expressed in Pichia pastoris). It was found that, when looking at the best 25 % of the clones, the clone ranking by online and offline methods matched in 83 % of the cases. With this approach a method for a primary phytase screening for the pre-selection of interesting clones was established. Furthermore, the optimization of 96-well MTP culture conditions were investigated. Optimal cultivation conditions such as shaking frequency, inoculation volume, initial glucose concentration and geometry of the micro titer plate were identified. The random mutagenesis library (epPCR library) of rXT-Phytase was generated and screening of rXT-Phytase library towards improved hydrolysis of InsPs was performed. Additional 1D 1H and 31P-NMR-spectra were acquired for the characterization of the selected variants. The chemical shifts together with the characteristic 1H1H and 1H31P-coupling patterns and coupling constants were used to identify the Inositol phosphates.