Sustainable and Continuous Synthesis of Enantiopure l‐Amino Acids by Using a Versatile Immobilised Multienzyme System

Abstract

The enzymatic synthesis of α-amino acids is a sustainable and efficient alternative to chemical processes, through which achieving enantiopure products is difficult. To more address this synthesis efficiently, a hierarchical architecture that irreversibly co-immobilises an amino acid dehydrogenase with polyethyleneimine on porous agarose beads has been designed and fabricated. The cationic polymer acts as an irreversible anchoring layer for the formate dehydrogenase. In this architecture, the two enzymes and polymer colocalise across the whole microstructure of the porous carrier. This multifunctional heterogeneous biocatalyst was kinetically characterised and applied to the enantioselective synthesis of a variety of canonical and noncanonical α-amino acids in both discontinuous (batch) and continuous modes. The co-immobilised bienzymatic system conserves more than 50 % of its initial effectiveness after five batch cycles and 8 days of continuous operation. Additionally, the environmental impact of this process has been semiquantitatively calculated and compared with the state of the art.

Characterization and evaluation of immobilized enzymes for applications in flow reactors

“…Based on these numbers, we conclude that engineering activity and stability tends to follow a parallel path, giving rise to cases that mainly occupy low/low and high/high activity/stability windows. Contrarily, we also find some few cases where the enzyme immobilization improves the reactor SY (activity) to higher extent than the enzyme TN (stability) -example k [45] -or vice versa -example o [27]. Nevertheless, the realistic evaluation of the operational stability (total turnover number) will require further studies that bring enzymes to their inactivation limits.…”

“…Hence, the internal surface of the packed material, the surface area generated during the monoliths manufacturing, and the inner area of microfluidic tubular reactors are decisive parameters for the enzyme loading. Most recent examples rest in the translation from batch reactors to PBRs using medium mesoporous or macroporous particles of diverse nature, such as cross-linked agarose, cross-linked polyacrylic polymers, and silica [18,[24][25][26][27][28]. The combination of medium-high protein loadings (10-100 mg/g), and dense packing into PBRs leads to a high catalyst concentration, and hence high STY.…”

Biocatalytic access to betazole using a one-pot multienzymatic system in continuous flow

“…1,4,20 Moreover, immobilising the different enzymes on the same intrapore bead surface improves further the performance of biocatalytic cascade systems due to facilitated intrapore diffusion of substrates and cofactors between different enzyme active sites. [42][43][44] Although co-immobilisation of multienzyme systems is highly challenging since there is no universal immobilisation chemistry or carrier, selective and optimal immobilisation of the different enzymes can be achieved by functionalization of the carrier surface with different reactive groups. 42,44,45 However, no examples have been reported on polymethacrylate-based carriers, which are relevant for continuous flow applications.…”

Selective Immobilization of Fluorescent Proteins for the Fabrication of Photoactive Materials

“…To that aim, we need to use a heterofunctional carrier that displays different reactive groups able to immobilize the different proteins through different chemistries. Although this concept has been previously exploited by our group [23,29,30,31], in this work we have genetically programmed two fluorescent proteins to be orthogonally co-immobilized on the same surface activated with two different highly selective groups for two different peptide tags. As proof of concept, Cys-sGFP and His-RFP were co-immobilized on agarose beads activated with disulfide and cobalt-chelate groups (AG-Co 2+ /S).…”