Controllably crosslinked dual enzymes enabled by genetic-encoded non-standard amino acid for efficiently enantioselective hydrogenation

Engineering ketoreductases for the enantioselective synthesis of chiral alcohols

“…The variants were subsequently crosslinked, directly from cell lysate, using a Cu-free bio-orthogonal click reaction with sym-dibenzo-1,5-cyclooctadiene-3,7-diyne 5 (Scheme 3). 45,46 The resulting CLEs exhibited improved catalytic efficiencies and thermal stabilities.…”

“…The resulting combi-CLEs of the KRED and GDH exhibited an enhanced activity in the synthesis of (R)-1-[3,5-bis (trifluoromethyl)phenyl] ethanol by reduction of the corresponding prochiral ketone. 45,46 Similarly, (S)-1-(2,6-dichloro-3fluorophenyl) ethanol was prepared in 76% yield and 99.99% ee, and the reusability of the combi-CLEs demonstrated. 48 Cross-linking, via cycloaddition of the azide functionalities with the cyclooctadiyne, is promoted by microwave irradiation and visible-light and affords an immobilised biocatalyst that is conveniently separated by centrifugation.…”

Designing an enzyme assembly line for green cascade processes using bio-orthogonal chemistry

“…Previous work showed that CLEAs produced using glutaraldehyde lost some of their original stereoselectivity with a reduced ee value of the product (72%). 74 This decrease in activity and enantioselectivity may result from the random and unstable nature of glutaraldehyde cross-linking 21 which results in disordered polymers 75 and unfavourable structural changes in the enzyme. 60 The simple one-step preparation and purification of O/S-CLEs using cell lysate supernatants preserves the enzyme activity observed with whole cells and avoids tedious purification.…”

“…36 Indeed, enzymatic reduction of prochiral ketones with highly engineered enzymes is the method of choice for the synthesis of enantiopure chiral alcohols as key building blocks for the industrial synthesis of a broad spectrum of pharmaceuticals. 37–39…”

Programmable Polyproteams of Tyrosine Ammonia Lyases as Cross-Linked Enzymes for Synthesizing p-Coumaric Acid

“…So far, immobilization technology based on physical adsorption [ 10 ] (such as entrapment [ 11 ]) and covalent attachment [ 12 , 13 , 14 ] (such as crosslinking [ 15 ]) has matured [ 16 ] and been widely applied in the fields of the pharmaceutical industry, food industry, and wastewater treatment. In fact, the ligation intensity between enzymes and their supporting carriers resulting from physical adsorption is far weaker than that from covalent attachment.…”