Asymmetric Reduction of Prochiral Ketones by Using Self‐Sufficient Heterogeneous Biocatalysts Based on NADPH‐Dependent Ketoreductases

Abstract

The development of cell-free and self-sufficient biocatalytic systems represents an emerging approach to address more complex synthetic schemes under nonphysiological conditions. Herein, we report the development of a self-sufficient heterogeneous biocatalyst for the synthesis of chiral alcohols without the need to add an exogenous cofactor. In this work, an NADPH-dependent ketoreductase was primarily stabilized and further co-immobilized with NADPH to catalyze asymmetric reductions without the addition of an exogenous cofactor. As a result, the immobilized cofactor is accessible, and thus, it is recycled inside the porous structure without diffusing out into the bulk, as demonstrated by single-particle in operando studies. This self-sufficient heterogeneous biocatalyst was used and recycled for the asymmetric reduction of eleven carbonyl compounds in a batch reactor without the addition of exogenous NADPH to achieve the corresponding alcohols in 100 % yield and >99 % ee; this high performance was maintained over five consecutive reaction cycles. Likewise, the self-sufficient heterogeneous biocatalyst was integrated into a plug flow reactor for the continuous synthesis of one model secondary alcohol, which gave rise to a space-time yield of 97-112 g L day ; additionally, the immobilized cofactor accumulated a total turnover number of 1076 for 120 h. This is one of the few examples of the successful implementation of continuous reactions in aqueous media catalyzed by cell-free and immobilized systems that integrate both enzymes and cofactors into the solid phase.

Deciphering the Effect of Microbead Size Distribution on the Kinetics of Heterogeneous Biocatalysts through Single-Particle Analysis Based on Fluorescence Microscopy

“…The image processing of time-lapse fluorescence microscopy experiments linked to mathematical modeling enables us to estimate apparent kinetic parameters at both single particle and sub-micrometric levels. These analyses were tested and validated using a new generation of self-sufficient heterogeneous biocatalysts recently developed in our group [27,28]. In these systems, the NAD(P)H-dependent alcohol dehydrogenases are co-immobilized with their corresponding cofactor on porous agarose microbeads.…”

Self-assembling all-enzyme hydrogels for biocatalytic flow processes

“…Indeed, diol product was formed for up to 4 hours indicating the effective retainment of the entrapped cofactor inside the hydrogel over 16 reactor column volumes. The obtained total turnover number of NADP(H) (TTNNADP(H)) was clearly in the economically feasible range 37 and even more then 3-fold higher than those recently reported for a self-sufficient heterogeneous biocatalyst, based on bead-bound ketoreductases with electrostatically co-immobilized NAD(P)H 38,39 . To the best of our knowledge, the TTNNADP(H) of >3400 observed here is the highest value ever reported for flow processes in devices lacking supportive membranes.…”

“…Conventional approaches address this issue by use of ultraand nanofiltration membrane technology or specifically modified surfaces that minimize or prevent cofactor loss through electrostatic attraction or even covalent immobilisation. While these approaches have led to increased TTNNADP(H) values, they are cost intensive and increase the complexity of production processes, thereby leading to limited economic viability 38,39,45,46 . Our self-assembly approach, in contrast, is straight-forward, scalable and, owing to the gel's intrinsic material properties, can be readily implemented in arbitrary reactor All rights reserved.…”

Chemoenzymatic Approaches to the Synthesis of the Calcimimetic Agent Cinacalcet Employing Transaminases and Ketoreductases

“…The resulting catalyst, defined as self‐sufficient heterogeneous biocatalyst, was able to catalyze asymmetric reductions without exogenous cofactor addition 18. Moreover, it could be successfully re‐used several batch operational cycles and operated in a plug‐flow reactor.…”