Mass-activated sorting of nanoliter droplets for label free screening of enzyme libraries
September 15-19, 2019
Directed Evolution is a key technology driving the utility of biocatalysis in pharmaceutical synthesis. Conventional approaches to Directed Evolution are conducted using bacterial cells expressing enzymes in microplates, with catalyzed reactions measured by HPLC, high-performance liquid chromatography-mass spectrometry (HPLC-MS), or optical detectors, which require either long cycle times or tailor-made substrates. To better ﬁt modern, fast-paced process chemistry development where solutions are rapidly needed for new substrates, droplet microﬂuidics interfaced with electrospray ionization (ESI)-MS provides a label-free high-throughput screening platform. To apply this method to industrial enzyme screening and to explore potential approaches that may further improve the overall throughput, we optimized the existing droplet−MS methods. Carryover between droplets, traditionally a signiﬁcant issue, was reduced to undetectable level by replacing the stainless steel ESI needle with a Teﬂon needle within a capillary electrophoresis (CE)−MS source. Throughput was improved to 3 Hz with a wide range of droplet sizes (10−50 nL) by tuning the sheath ﬂow within the CE−MS source. The optimized method was demonstrated by screening reactions using two diﬀerent transaminase libraries. Good correlations (r2 ∼ 0.95) were found between the droplet−MS and LC−MS methods, with 100% match on hit variants. We further explored the capability of the system by performing in vitro transcription−translation inside the droplets and directly analyzing the intact reaction mixture droplets by MS. The synthesized protein attained comparable activity to the protein standard, and the complex samples appeared well tolerated by the MS. The success of the above applications indicates that the MS analysis of the microﬂuidic droplets is an available option for considerably accelerating the screening of enzyme evolution libraries.
Jeff Moore, "Mass-activated sorting of nanoliter droplets for label free screening of enzyme libraries" in "Enzyme Engineering XXV", Huimin Zhao, University of Illinois at Urbana-Champaign, USA John Wong, Pfizer, USA Eds, ECI Symposium Series, (2019). https://dc.engconfintl.org/enzyme_xxv/131