Development of a High-Throughput Screening Assay for Carboligase Activity

3 Development of a High-Throughput Screening Assay for Carboligase Activity 

Growth conditions in deep-well microtiter plates were optimized with respect to optimal expression of active enzymes (Fig. 2.2.1.1). The best results were obtained with an expression time of 20 h at 37 °C (Fig. 2.2.1.1, lanes 7-9). Subsequently, E. coli cells were enzymatically disrupted by lysozyme treatment, and the carboligase activity was monitored by a modified tetrazolium salt color assay [16], This color assay is based on the reduction of the 2,3,5-triphenyltetrazolium chloride (TTC) 13 to the corresponding formazan 15, which has an intense red color (Fig. 2.2.1.2A). Before screening ofa BFD variant library, substrates and products were tested in the color assay. Neither substrate, benzaldehyde 4 nor dimethoxy-acetaldehyde 8, reduced TTC 13 however, the product 2-hydroxy-3,3-dimethoxy-propiophenone 10 already caused color formation at low concentrations of 2.5-10 mM (Fig. 2.2.1.2B). Benzoin 12 as the product also gave a color change at a similar concentration (data not shown). 

The expression cultures were induced (0 h) and grown for 3 and 20 h. Cell extracts (corresponding to ODsgo = 0.15 of the bacterial culture) were harvested by centrifugation, and the proteins were separated on a 12% (w/v) polyacrylamide gel, and stained with Coomassie brilliant blue. 

Additionally, we tested cell extracts in the colorimetric assay both negative control strains E. coli pET28a containing the empty vector and E. coli BFDL476Q (Table 2.2.1.2) did not show any enzyme activity in the TTC assay, whereas E. coli BAL (Table 2.2.1.2) led to the formation of an intense red color (data not shown). 

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