Journal
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
Volume 56, Issue 9, Pages 2296-2301Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.201610821
Keywords
biocatalysis; continuous flow; multi-step transformations; protein purification; thin films
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Funding
- Taihi Hong Memorial award
- Australian Research Council
- Government of South Australia
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Nature applies enzymatic assembly lines to synthesize bioactive compounds. Inspired by such capabilities, we have developed a facile method for spatially segregating attached enzymes in a continuous-flow, vortex fluidic device (VFD). Fused His(n)-tags at the protein termini allow rapid bioconjugation and consequent purification through complexation with immobilized metal affinity chromatography (IMAC) resin. Six proteins were purified from complex cell lysates to average homogeneities of 76%. The most challenging to purify, tobacco epi-aristolochene synthase, was purified in only ten minutes from cell lysate to near homogeneity (>90%). Furthermore, this reaction-ready system demonstrated excellent stability during five days of continuous-flow processing. Towards multi-step transformations in continuous flow, proteins were arrayed as ordered zones on the reactor surface allowing segregation of catalysts. Ordering enzymes into zones opens up new opportunities for continuous-flow biosynthesis.
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