Fig 5 - uploaded by Javier Rocha Martín
Content may be subject to copyright.
Reaction course and TTN of ethyl acetoacetate asymmetric reduction by AG-G@Tt27-HBDH(PEI) with soluble or co-immobilised NADH. (A) 10% load of Tt27-HBDH immobilised biocatalyst with (red circles) or without (grey squares) co-immobilised NADH as well as soluble Tt27-HBDH (1.1 mg mL −1 ) (blue triangles) were incubated with 10 mM EAA, 5% 2-propanol (0.66 M), 1 mM NADH in 10 mM Tris-HCl at pH 8.0, for 24 h at 25 °C with orbital agitation and a total reaction volume of 5 mL. AG-G carrier without enzyme (green diamonds) incubated under the same conditions was used as control. (B) Product yield and total turnover number during 10 cycles of 24 h batch reactions at 25 °C. Chromatographic yield (%) of the asymmetric reduction of EAA catalysed by AG-G@Tt27-HBDH (PEI) with (green squares) or without (dark blue circles) co-immobilised NADH in consecutive batch reactions. Reactions were carried out as in panel A. Green bars represent the cofactor accumulated TTN after each consecutive batch reaction cycle. Blue bars represent the singlereaction cofactor TTN using soluble cofactor.
Source publication
β-Hydroxyesters are essential building blocks utilised by the pharmaceutical and food industries in the synthesis of functional products. Beyond the conventional production methods based on chemical catalysis or whole-cell synthesis, the asymmetric reduction of β-ketoesters with cell-free enzymes is gaining relevance. To this end, a novel thermophi...
Contexts in source publication
Context 1
... Michaelis-Menten kinetic parameters of Tt27-HBDH were determined towards the most active substrates in both oxidative and reductive directions (Table 2 and Fig. S5 †). While the k cat values fall in the range reported for other HBDHs, the K M values towards both ethyl β-keto-and hydroxy-esters are significantly higher than the corresponding values reported for other members of this family. Since this family of enzymes accepts acetoacetyl-CoA and 3-hydroxybutyryl-CoA as natural substrates, ...
Context 2
... demonstrated the functionality of the immobilised Tt27-HBDH towards the NADH absorbed to the PEI layer of the heterogeneous biocatalyst, we performed the asymmetric reduction of EAA without supplying exogenous cofactor in batch. Fig. 5A shows that the co-immobilization of the enzyme and the cofactor increases both the reaction rate and the product yield after 24 h, when compared to the immobilised enzyme using exogenous cofactor. Expectedly, using both soluble enzyme and cofactor, we observed a higher synthetic rate likely due to the 70% activity reduction suffered ...
Context 3
... enzyme and the cofactor increases both the reaction rate and the product yield after 24 h, when compared to the immobilised enzyme using exogenous cofactor. Expectedly, using both soluble enzyme and cofactor, we observed a higher synthetic rate likely due to the 70% activity reduction suffered upon the enzyme immobilization and the PEI coating (Fig. 5A). However, the soluble system is hard to reuse after one cycle; on the contrary, the heterogeneous biocatalysts herein utilised are easily separated from the reaction products through vacuum filtration and reused for consecutive discontinuous reaction cycles. Immobilised Tt27-HBDH using both co-immobilised and soluble cofactor were ...
Context 4
... the heterogeneous biocatalysts herein utilised are easily separated from the reaction products through vacuum filtration and reused for consecutive discontinuous reaction cycles. Immobilised Tt27-HBDH using both co-immobilised and soluble cofactor were re-used for up to 10 cycles maintaining more than 60% product conversion after each 24 h cycle (Fig. 5B). ...
Citations
... 29 The physicochemical uniqueness of the histidines makes them an excellent residue for the surface engineering of enzymes to tune the enzyme orientation during the immobilization process. In this work, we chose two model dehydrogenases, one from Bacillus stearothermophilus (BsADH) 32 and another from Thermus thermophilus (TtHBDH), 33 two relevant enzymes in applied biocatalysis. We engineered these enzymes with His-clusters in different regions. ...
... To further demonstrate the generality of this approach in another enzyme, we selected the (S)-3-hydroxybutyryl-CoA dehydrogenase from T. thermophilus (TtHBDH), 33 a hexametric enzyme of 31.8 kDa/ subunit, and engineered its surface with His-clusters. Using an enzyme structural model based on the template 3-hydroxybutyryl-CoA dehydrogenase from Clostridium acetobutylicum (PDB: 6ACQ; 54% homology) previously created by our group, 33 we followed a similar workflow as the one followed for BsADH. After performing COREX/Best analysis of this model ( Figure 7A), we proposed introducing up to 4 His residues in the positions R99, R129, R130, and R250. ...
... This was possible due to the thermophilic origin of TtHBDH, which was purified by thermal shock. 33 Once cloned, all variants were expressed, semipurified, and structurally and functionally characterized (Table S6 and Figure S9). The His-clustered variants had 2−4 times lower specific activities than the untagged variant, nevertheless with sufficient activity to perform the immobilization experiments. ...
Enzyme immobilization is a key enabling technology for a myriad of industrial applications, yet immobilization science is still too empirical to reach highly active and robust heterogeneous biocatalysts through a general approach. Conventional protein immobilization methods lack control over how enzymes are oriented on solid carriers, resulting in negative conformational changes that drive enzyme deactivation. Site-selective enzyme immobilization through peptide tags and protein domains addresses the orientation issue, but this approach limits the possible orientations to the N- and C-termini of the target enzyme. In this work, we engineer the surface of two model dehydrogenases to introduce histidine clusters into flexible regions not involved in catalysis, through which immobilization is driven. By varying the position and the histidine density of the clusters, we create a small library of enzyme variants to be immobilized on different carriers functionalized with different densities of various metal chelates (Co²⁺, Cu²⁺, Ni²⁺, and Fe³⁺). We first demonstrate that His-clusters can be as efficient as the conventional His-tags in immobilizing enzymes, recovering even more activity and gaining stability against some denaturing agents. Furthermore, we find that the enzyme orientation as well as the type and density of the metal chelates affect the immobilization parameters (immobilization yield and recovered activity) and the stability of the immobilized enzymes. According to proteomic studies, His-clusters enable a different enzyme orientation as compared to His-tag. Finally, these oriented heterogeneous biocatalysts are implemented in batch reactions, demonstrating that the stability achieved by an optimized orientation translates into increased operational stability.
... The versatility of this methodology was demonstrated by coating two sets of MNPs that presents different sizes (12 vs. 30 nm) and shapes (spheroids vs. cubes) with three different TM coatings (Co 2+ , Ni 2+ , and Cu 2+ ). The efficiency of each combination of magnetic core and TM inorganic coating was tested in comparison to NTA-TM coated MNPs, using the monomeric superfolder Green Fluorescent Protein (sGFP) and the tetrameric alcohol dehydrogenases from Bacillus stearothermophilus (BsADH) harboring the 6x-His-tag at their N-terminus [18], and 3-hydroxyacyl-CoA dehydrogenase from Thermus thermophilus (TtHBDH) whose C-terminus is fused to a 6xHis-tag [19]. These model His-tagged proteins were selected for their biotechnological interest, but also due their difference in its oligomeric state, which affects the total number of His-tags per enzyme molecule. ...
... Enantiopure alcohols as valuable synthons are widely employed in pharmaceuticals as drug intermediates [1]. Among them, ethyl (R)-3-hydroxybutyrate ((R)-EHB) is a vital chiral intermediate used in the production of many drugs, such as β-lactamase inhibitors and anti-glaucoma drugs [2,3]. As an eco-friendly and sustainable technology, biocatalysis has been widely used in the synthesis of pharmaceuticals [4,5]. ...
Ionic liquids (ILs) which synthesized from bio-renewable materials have recently attracted much attention for their applications in biocatalysis. Ethyl (R)-3-hydroxybutyrate ((R)-EHB) as a versatile chiral intermediate is of great interest in pharmaceutical synthesis. This study focuses on evaluating the performances of choline chloride (ChCl)-based and tetramethylammonium (TMA)-based neoteric ILs in the efficient synthesis of (R)-EHB via the bioreduction of ethyl acetoacetate (EAA) at high substrate loading by recombinant Escherichia coli cells. It was found that choline chloride/glutathione (ChCl/GSH, molar ratio 1:1) and tetramethylammonium/cysteine ([TMA][Cys], molar ratio 1:1) as eco-friendly ILs not only enhanced the solubility of water-insoluble EAA in the aqueous buffer system, but also appropriately improved the membrane permeability of recombinant E. coli cells, thus boosting catalytic reduction efficiency of EAA to (R)-EHB. In the developed ChCl/GSH- or [TMA][Cys]-buffer systems, the space-time yields of (R)-EHB achieved 754.9 g/L/d and 726.3 g/L/d, respectively, which are much higher than neat aqueous buffer system (537.2 g/L/d space-time yield). Meanwhile, positive results have also been demonstrated in the bioreduction of other prochiral ketones in the established IL-buffer systems. This work exhibits an efficient bioprocess for (R)-EHB synthesis under 325 g/L (2.5 M) substrate loading, and provides promising ChCl/GSH- and [TMA][Cys]-buffer systems employed in the biocatalysis for hydrophobic substrate.
... Cofactor recycling is necessary for the future application of the formulation at the industrial level. A self-sufficient heterogeneous biocatalyst system has been reported to successfully realize cofactor recycling in aqueous media and maintain high catalytic performance over consecutive reaction cycles without the addition of exogenous cofactors [46,6,63]. In this system, enzymes are first immobilized on a porous support. ...
Patulin is one of the most important mycotoxins that contaminates fruit-derived products and causes acute or chronic toxicity in humans. In the present study, a novel patulin-degrading enzyme preparation was developed by taking a short-chain dehydrogenase/reductase and covalently linking it to dopamine/polyethyleneimine co-deposited magnetic Fe3O4 particles. Optimum immobilization provided 63% immobilization efficiency and 62% activity recovery. Moreover, the immobilization protocol substantially improved thermal and storage stabilities, proteolysis resistance, and reusability. Using reduced nicotinamide adenine dinucleotide phosphate as a cofactor, the immobilized enzyme exhibited a detoxification rate of 100% in phosphate-buffered saline and a detoxification rate of more than 80% in apple juice. The immobilized enzyme did not cause adverse effects on juice quality and could be magnetically separated quickly after detoxification to ensure convenient recycling. Moreover, it did not exhibit cytotoxicity against a human gastric mucosal epithelial cell line at a concentration of 100 mg/L. Consequently, the immobilized enzyme as a biocatalyst had the characteristics of high efficiency, stability, safety, and easy separation, establishing the first step in building a bio-detoxification system to control patulin contamination in juice and beverage products.
... López-Gallego and coworkers reported a self-sufficient heterogeneous biocatalyst by coimmobilising both enzyme and cofactor on glyoxyl-agarose macroporous beads (Orrego et al., 2021). After covalent immobilisation of a thermophilic (S)-2hydroxybutyryl-CoA dehydrogenase from Thermus thermophilus HB27 (Tt27-HBDH), the remaining formyl groups on the carrier allowed for a subsequent coating with polyethyleneimine (PEI) to give a cationic layer in which NADH molecules could be embedded and reversibly immobilised through ionic interactions. ...
Alcohol dehydrogenases (ADHs) have become important catalysts for stereoselective oxidation and reduction reactions of alcohols, aldehydes and ketones. The aim of this contribution is to provide the reader with a timely update on the state-of-the-art of ADH-catalysis. Mechanistic basics are presented together with practical information about the use of ADHs. Current concepts of ADH engineering and ADH reactions are critically discussed. Finally, this contribution highlights some prominent examples and future-pointing concepts.
... In this heterogeneous biocatalyst, both enzyme and cofactor are confined within the same porous space, but the reversible binding of NADH lets it travel between the active sites of the irreversibly bound enzymes. The system showed high operational stability in a repeated batch process, as well as high stability under high temperatures and acid pH [53]. An interesting approach to localize two enzymes in different compartments, allowing tandem reactions, is the use of a coreeshell of chitosan-coated alginate beads [54]. ...
Despite their sustainability, the potential of biocatalytic processes in industrial production is far from being realized. The main challenges in this field are the development of highly active, robust, and stable biocatalysts, the efficient regeneration of cofactors, and the prevention of biocatalyst deactivation under harsh industrial conditions. In addition to biocatalyst engineering, efficient enzyme and cell immobilization plays a crucial role in process feasibility. Reactor miniaturization, continuous operation, and integration with in situ product removal, process analytics, and cascade reactions that reduce the number of process steps enable process intensification. Mathematical model-based reactor and process design comprising time-scale analysis, and efficient capacity increase can push the boundaries of biocatalytic processes toward industrial requirements. This review highlights the latest trends in efficient biocatalyst immobilization, miniaturization, and process integration to intensify biocatalytic processes.
Developing two self-sufficient heterogeneous biocatalysts to synthesise enantiopure β-hydroxy esters in continuous flow. Operational conditions are tuned to maximise the productivity and stability of biocatalytic packed-bed reactors.
Biocatalyst heterogenisation may enable robust processes that can be applied in biorefineries to selectively valorise highly functionalised platform chemicals. In this work, we co-immobilise two dehydrogenases and successfully apply them in the selective reduction of 5-hydroxymethylfurfural (HMF) to 2,5-bis(hydroxymethyl) furan (BHMF) with efficient in situ cofactor regeneration. First, we select the best enzyme candidates (an alcohol dehydrogenase from Escherichia coli together with a thermostable glucose dehydrogenase from Bacillus subtilis) and then screen a variety of carriers and chemistries to find the optimal individual immobilisation protocols for each dehydrogenase. As a result, methacrylate carriers (Purolite™) functionalised with either aldehydes or with epoxy and cobalt-chelate groups co-immobilise both enzymes in high yields with a sufficient activity recovery (>20%). These optimal heterogeneous biocatalysts enable the quantitative bio-reduction of HMF to BHMF with >99% selectivity in only fifteen minutes, exhibiting an outstanding reusability of >15 batch cycles with a total volumetric productivity of ∼5 g L⁻¹ h⁻¹ of BHMF. Preliminary experiments on a semipreparative scale with HMF loadings of 40 mM also reach high product conversions (86%). Overall, the judicious selection of enzymes, carriers and reaction conditions enables the design of robust biocatalysts that may contribute to paving the way to the valorisation of highly functionalised chemicals in biorefineries.
