Figure 2

The best activity of cells expressing PAMO is obtained with mid log cells, induced for PAMO expression for 4 hours and without external addition of riboflavin. (A) Analysis of the best cellular growth stage for the induction of PAMO expression. Top10 cells were grown to mid log, late log or stationary phase as described in the Methods section, harvested and grown in the presence of 0.2% L-arabinose in 96-sdMTP for 3 hours at 30°C. Following PAMO expression, cells were used for the bioconversion of phenylacetone in 96-sdMTP as described in the legend to Figure 1. (B) Assessment of the best induction time for the production of PAMO. Top 10 cells were grown to mid log phase and used for the production of PAMO in 96-sdMTP as described in the legend to Figure 2A. Starting 2 hours after induction, samples were removed hourly and used for the bioconversion of phenylacetone in 96-sdMTP for 2 hours at 37°C followed by the analysis of the benzyl acetate content. (C) Effect of external riboflavin addition. Mid log Top10 cells were induced for PAMO expression as described in the legend to Figure 2A in the presence of the indicated amounts of riboflavin. Following PAMO expression, cells were used for the bioconversion of phenylacetone as described in the legend to Figure 1 followed by the analysis of the benzyl acetate content. All experiments were performed in duplicate and the values obtained were within 5% of each other.