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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*Rh-III-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.

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., More accurate library screening: A new high‐throughput screening platform including affinity purification has been developed based on an in vitro screening approach for MBP‐tagged nitrobindin having a Cp*RhIII complex as an artificial cofactor. Using this platform for directed evolution, a biohybrid catalyst with clearly enhanced activity toward the cycloaddition of acetophenone oximes with alkynes was obtained.

A unique π-expanded reaction cavity tethering a polycyclic moiety which provides a platform for substrate binding was constructed within the robust β-barrel structure of nitrobindin (NB). NB variants with cavities of different sizes and shapes are coupled with N-(1-pyrenyl)maleimide (Pyr) to prepare a series of NB-Pyr conjugates. The orientation of the pyrene moiety is fixed within the cavity by the coupling reaction. The fluorescent quenching analysis of NB-Pyr indicates that azachalcone (aza), which is a dienophile for a Diels-Alder (DA) reaction, is efficiently incorporated within the pyrene-linked reaction cavity by the aromatic interaction. The DA reaction between aza and cyclopentadiene proceeds within the reaction cavity of NB-Pyr in the presence of CuII ion in high yield and high enantio- and regioselectivity.

Pulsating compressive fatigue tests were conducted by using single-edge-notched specimens of a low-carbon steel under in-plane four-point bending. The development of crack growth was studied both experimentally and theoretically. Under cyclic compression, a crack initiated at the notch root, and then stopped at a certain crack length. The relation between the crack growth rate and the effective stress intensity range for short cracks at notches was identical to that obtained for long cracks. The crack opening stress calculated from the stress distribution at the applied minimum stress agreed with the experimental data except near the threshold. A method was proposed to estimate the upper bound of the non-propagating crack length.