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Rha, Eugene,Kim, Sujin,Choi, Su‐,Lim,Hong, Seung‐,Pyo,Sung, Moon‐,Hee,Song, Jae J.,Lee, Seung‐,Goo Blackwell Publishing Ltd 2009 FEBS JOURNAL Vol.276 No.21
<P>The tyrosine phenol‐lyase from <I>Symbiobacterium toebii</I> was engineered to improve both its stability and catalytic activity by the application of random mutagenesis and subsequent reassembly of the acquired mutations. Activity screening of the random library produced four mutants with a two‐fold improved activity, whereas parallel screening after heat treatment at 65 °C identified three mutants with half‐inactivation temperatures improved by up to 5.6 °C. The selected mutants were then reassembled using the staggered extension PCR method, and subsequent screening of the library produced seven mutants with up to three‐fold improved activity and half‐inactivation temperatures improved by up to 11.2 °C. Sequence analyses revealed that the stability‐improved hits included A13V, E83K and T407A mutations, whereas the activity‐improved hits included the additional T129I or T451A mutation. In particular, the A13V mutation was propagated in the hits with improved stability during the reassembly–screening process, indicating the critical nature of the N‐terminal moiety for enzyme stability. Furthermore, homology modeling of the enzyme structure revealed that most of the stability mutations were located around the dimer–dimer interface, including the N‐terminus, whereas the activity‐improving mutations were located further away, thereby minimizing any interference that would be detrimental to the co‐improvement of the stability and catalytic activity of the enzyme.</P>
Mesh-integrated microdroplet array for simultaneous merging and storage of single-cell droplets.
Um, Eujin,Rha, Eugene,Choi, Su-Lim,Lee, Seung-Goo,Park, Je-Kyun Royal Society of Chemistry 2012 Lab on a chip Vol.12 No.9
<P>We constructed a mesh-grid integrated microwell array which enables easy trapping and consistent addition of droplets. The grid acts as a microchannel structure to guide droplets into the microwells underneath, and also provides open access for additional manipulation in a high-throughput manner. Each droplet in the array forms a stable environment of pico-litre volume to implement a single-cell-based assay.</P>