Directed Evolution of a Cp*RhIII-Linked Biohybrid Catalyst Based on a Screening Platform with Affinity Purification |
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| Authors: | Shunsuke Kato Prof. Akira Onoda Naomasa Taniguchi Prof. Ulrich Schwaneberg Prof. Takashi Hayashi |
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| Affiliation: | 1. Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871 Japan;2. Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen, 52074 Germany |
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| Abstract: | Directed evolution of Cp*RhIII-linked nitrobindin (NB), a biohybrid catalyst, was performed based on an in vitro screening approach. A key aspect of this effort was the establishment of a high-throughput screening (HTS) platform that involves an affinity purification step employing a starch-agarose resin for a maltose binding protein (MBP) tag. The HTS platform enables efficient preparation of the purified MBP-tagged biohybrid catalysts in a 96-well format and eliminates background influence of the host E. coli cells. Three rounds of directed evolution and screening of more than 4000 clones yielded a Cp*RhIII-linked NB(T98H/L100K/K127E) variant with a 4.9-fold enhanced activity for the cycloaddition of acetophenone oximes with alkynes. It is confirmed that this HTS platform for directed evolution provides an efficient strategy for generating highly active biohybrid catalysts incorporating a synthetic metal cofactor. |
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| Keywords: | artificial metalloenzymes biohybrid catalysts C−H bond functionalization directed evolution rhodium |
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