Catch-and-Release: The Assembly, Immobilization, and Recycling of Redox-Reversible Artificial Metalloenzymes

Abstract

Technologies to improve the applicability of artificial metalloenzymes (ArMs) are gaining considerable interest; one such approach is the immobilization of these biohybrid catalysts on support materials to enhance stability and enable their retention, recovery, and reuse. Here, we describe the immobilization of polyhistidine-tagged ArMs that allow the redox-controlled replacement of catalytic cofactors that have lost activity, e.g., due to poisoning or decomposition, on immobilized metal affinity chromatography resins. By using periplasmic siderophore-binding protein scaffolds that originate from thermophilic bacteria ( Gst CeuE and Pth CeuE) in combination with a siderophore-linked imine reduction catalyst, reaction rates were achieved that are about 3.5 times faster than those previously obtained with Cj CeuE, the analogous protein of Campylobacter jejuni. Upon immobilization, the Gst CeuE-derived ArM showed a decrease in turnover frequency in the reduction of dehydrosalsolidine by 3.4-fold, while retaining enantioselectivity (36%) and showing improved stability that allowed repeat recovery and recycling cycles. Catalytic activity was preserved over the initial four cycles. In subsequent cycles, a gradual reduction of activity was evident. Once the initial activity decreased to around 40% of the initial activity (23rd recycling cycle), the redox-triggered artificial cofactor release permitted the subsequent recharging of the immobilized protein scaffold with fresh, active cofactor, thereby restoring the initial catalytic activity of the immobilized ArM and allowing its reuse for several more cycles. Furthermore, the ArM could be assembled directly from protein present in crude cell extracts, avoiding time-consuming and costly protein purification steps. Overall, this study demonstrates that the immobilization of redox-reversible ArMs facilitates their “catch-and-release” assembly and disassembly and the recycling of their components, improving their potential commercial viability and environmental footprint.

Redox-reversible siderophore-based catalyst anchoring within cross-linked artificial metalloenzyme aggregates enables enantioselectivity switching

“…1(b). This represents a significant advance over prior studies that utilised an IMAC resin as carrier material for immobilisation, 15 in which both free and immobilised ArM were only stable between pH 6 and 7. The formed Gst- 1 -CLArMAs microparticles exihibited a near neutral zeta potential of 0.3 ± 3.4 mV (Fig.…”

“…We have recently reported an affinity-based approach to immobilise a histidine-tagged redox-reversible artificial metalloenzyme onto conventional immobilised metal affinity chromatography (IMAC) resins. 15 Our system takes advantage of the reversible binding of a synthetic catalyst via an Fe III -siderophore anchoring strategy 2 to the protein scaffold, thereby enabling catalyst release and the subsequent recovery of the individual components. This catch-and-release strategy successfully enabled the immobilisation of the ArMs and their direct assembly from crude cell lysates.…”

“…Partial detachment of the catalyst during agglomeration and cross-linking offers a plausible explanation for the iridium losses in the final samples. With a K d value of 9.9 ± 0.9 nM, 15 Gst has only a moderate binding affinity for 1 . Therefore, the immobilisation yield, based on catalytic centres, is approximately 65%.…”

“…This represents a significant improvement over the previously-reported IMAC resin-supported Gst- 1 , where immobilisation in Ni-sepharose microbeads led to a 3.4-fold decrease in TOF. 15…”

“…Here we report the immobilisation of our previously-reported artificial imine reductase Gst- 1 -ArM (Scheme 1(a)). 15 Gst- 1 -ArM consists of an iridium-containing catalyst attached to an Fe III -azotochelin-based anchor ( 1 ), which binds strongly but reversibly to Gst, a periplasmic binding protein scaffold from Geobacillus stearothermophilus . 25 Gst- 1 -ArM agglomerates in the presence of organic solvents or ammonium sulfate (Scheme 1(b)), and the high density of lysine residues on the surface of Gst (Scheme 1(a)) enabled subsequent cross-linking with glutaraldehyde (Scheme 1(c)).…”