High-Throughput Screening for Carboligation Activity with the Substrates Benzaldehyde and Dimethoxyacetaldehyde

5 High-Throughput Screening for Carboligation Activity with the Substrates Benzaldehyde and Dimethoxyacetaldehyde 

The mutated BED genes were cloned into the vector pET28a (Table 2.2.1.2) and the resulting plasmids were transformed into E.coli BL21 (DEB) (Table 2.2.1.2). 

The clones were transferred into 96 deep-well microtiter plates filled with 100 pL LB medium (10 g NaCl, 5g yeast extract, 10 g trypton) supplemented with 50 pg mL kanamycin. After overnight shaking (600 rpm) at 37 °C, 600 pL LB medium containing kanamycin (50 pg mL ) were added. After an additional 

Biosciences, Uppsala, Sweden). The purification was performed as described elsewhere [11]. Finally, the purified enzyme variants were lyophilized and stored at -20 C. 

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