Enzyme engineering for pharmaceutical process chemistry and manufacturing has become a revolutionary technology. Conventional approaches to enzyme evolution are conducted using cells in microplates, coupled to either HPLC-MS or optical detection, which requires long screening time, high consumption of the reagents, or tailor made surrogate substrate. To be more compatible with modern fast-pace process chemistry development where solutions are rapidly needed for any substrate, we are developing a system to reduce reagent use, eliminate surrogate substrates, and improve overall throughput in enzyme engineering and screening.<br/>The system consists of cell free protein synthesis, droplet microfluidics, and mass spectrometry. The enzyme variants were synthesized using PURExpress in vitro transcription translation (ivTT) kit. The total time required for protein synthesis is 2 hours, and the total reaction volume is 10 uL. Following protein engineering, 1 uL of the substrate solution was added to the ivTT mixture and the enzymatic reaction was conducted for 4 hours. Once completed, the enzymatic reaction mixture was diluted with 20% ACN, which was than segmented by oil (perfluorodecalin) and directly infused to MS. Currently, the reactions were performed in 96 well microplate, after the ACN dilution, a thin layer of oil was placed on top of the aqueous solution in each well as well as on top of the whole plate. Then, a computer numerical control machine and a syringe pump were used to generate the water/oil droplets with a droplet size of 50 nL. The droplets were stored in a 250 nm i.d. Teflon tubing, which were then infused into the MS using the same syringe pump. The entire assay time for a 96 well plate with 4 replicates for each sample was ~2 min, corresponding to a sampling rate of 3 samples per second, which significantly improved the throughput of the enzyme screening process.<br/>In the future, this method will be transferred to a microfluidic device, where the ivTT reaction and the enzymatic reaction will all be integrated on one single device, so that the throughput of the entire enzyme evolution process will be further improved.

Droplets microfluidic platform for high throughput enzyme screening

253rd National Meeting (2017)

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