1. Directed Evolution of a Cp*Rh III -Linked Biohybrid Catalyst Based on a Screening Platform with Affinity Purification.
- Author
-
Kato S, Onoda A, Taniguchi N, Schwaneberg U, and Hayashi T
- Subjects
- Catalysis, Organometallic Compounds chemistry, Ruthenium Compounds chemistry, Chromatography, Affinity methods, Chromatography, Agarose methods, High-Throughput Screening Assays methods, Maltose-Binding Proteins metabolism, Organometallic Compounds metabolism, Ruthenium Compounds metabolism, Starch chemistry
- Abstract
Directed evolution of Cp*Rh
III -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., (© 2020 The Authors. ChemBioChem published by Wiley-VCH GmbH.)- Published
- 2021
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