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•Xys1Δ immobilized on activated Relizyme R403/S-glyoxyl was selected to set up a PRB for the continuous production of XOS.•The highest specific productivity for XOS (3,277 gXOS ...L−1 h−1) was obtained at 10 mL/min flow rate.•This value was 14-fold higher than the specific productivity value obtained in batch mode.•The PBR packed with R403S-G-Xys1Δ biocatalyst was continuously operated for 120 h.
The present study focuses on the development and optimization of a packed-bed reactor (PBR) for continuous production of xylooligosaccharides (XOS) from xylan. For this purpose, three different methacrylic polymer-based supports (Relizyme R403/S, Purolite P8204F and Purolite P8215F) activated with glyoxyl groups were morphologically characterized and screened for the multipoint covalent immobilization of a xylanase. Based on its physical and mechanical properties, maximum protein loading and thermal stability, Relizyme R403/S was selected to set up a PRB for continuous production of XOS from corncob xylan. The specific productivity for XOS at 10 mL/min flow rate was 3277 gXOS genzyme−1 h−1 with a PBR. This PBR conserved >90% of its initial activity after 120 h of continuous operation.
Glycosylation is one of the most efficient biocompatible methodologies to enhance the water solubility of natural products, and therefore their bioavailability. The excellent regio- and ...stereoselectivity of nucleotide sugar-dependent glycosyltransferases enables single-step glycosylations at specific positions of a broad variety of acceptor molecules without the requirement of protection/deprotection steps. However, the need for stoichiometric quantities of high-cost substrates, UDP-sugars, is a limiting factor for its use at an industrial scale. To overcome this challenge, here we report tailor-made coimmobilization and colocalization procedures to assemble a bi-enzymatic cascade composed of a glycosyltransferase and a sucrose synthase for the regioselective 5-O-β-D-glycosylation of piceid with in situ cofactor regeneration. Coimmobilization and colocalization of enzymes was achieved by performing slow immobilization of both enzymes inside the porous support. The colocalization of both enzymes within the porous structure of a solid support promoted an increase in the overall stability of the bi-enzymatic system and improved 50-fold the efficiency of piceid glycosylation compared with the non-colocalized biocatalyst. Finally, piceid conversion to resveratrol 3,5-diglucoside was over 90% after 6 cycles using the optimal biocatalyst and was reused in up to 10 batch reaction cycles accumulating a TTN of 91.7 for the UDP recycling.
•PEI-coated supports promote higher stability and strong adsorption of the enzymes.•UGT immobilized on Ag-PEI was 36-fold more stable than the soluble form.•SuSyAc immobilized on Ag-PEI was 480-fold more stable than soluble SuSyAc.•Colocalization of both enzymes enhanced 50-fold the glycosylation rate of piceid.•After 10 batch reaction cycles, the accumulated TTN for the UDP recycling was 91.7.
Immobilized enzymes have a very large region that is not in contact with the support surface and this region could be the target of new stabilization strategies. The chemical amination of these ...regions plus further cross-linking with aldehyde-dextran polymers is proposed here as a strategy to increase the stability of immobilized enzymes. Aldehyde-dextran is not able to react with single amino groups but it reacts very rapidly with polyaminated surfaces. Three lipases-from
(TLL),
(RML), and
B (CALB)-were immobilized using interfacial adsorption on the hydrophobic octyl-Sepharose support, chemically aminated, and cross-linked. Catalytic activities remained higher than 70% with regard to unmodified conjugates. The increase in the amination degree of the lipases together with the increase in the density of aldehyde groups in the dextran-aldehyde polymer promoted a higher number of cross-links. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of those conjugates demonstrates the major role of the intramolecular cross-linking on the stabilization of the enzymes. The highest stabilization was achieved by the modified RML immobilized on octyl-Sepharose, which was 250-fold more stable than the unmodified conjugate. The TLL and the CALB were 40-fold and 4-fold more stable than the unmodified conjugate.
Enzyme immobilization by multipoint covalent attachment on supports activated with aliphatic aldehyde groups (e.g., glyoxyl agarose) has proven to be an excellent immobilization technique for enzyme ...stabilization. Borohydride reduction of immobilized enzymes is necessary to convert enzyme–support linkages into stable secondary amino groups and to convert the remaining aldehyde groups on the support into hydroxy groups. However, the use of borohydride can adversely affect the structure–activity of some immobilized enzymes. For this reason, 2-picoline borane is proposed here as an alternative milder reducing agent, especially, for those enzymes sensitive to borohydride reduction. The immobilization-stabilization parameters of five enzymes from different sources and nature (from monomeric to multimeric enzymes) were compared with those obtained by conventional methodology. The most interesting results were obtained for bacterial (R)-mandelate dehydrogenase (ManDH). Immobilized ManDH reduced with borohydride almost completely lost its catalytic activity (1.5% of expressed activity). In contrast, using 2-picoline borane and blocking the remaining aldehyde groups on the support with glycine allowed for a conjugate with a significant activity of 19.5%. This improved biocatalyst was 357-fold more stable than the soluble enzyme at 50 °C and pH 7. The results show that this alternative methodology can lead to more stable and active biocatalysts.
We present the synthesis, photophysical properties, and biological application of nontoxic 3-azo-conjugated BODIPY dyes as masked fluorescent biosensors of hypoxia-like conditions. The synthetic ...methodology is based on an operationally simple NN bond-forming protocol, followed by a Suzuki coupling, that allows for a direct access to simple and underexplored 3-azo-substituted BODIPY. These dyes can turn on their emission properties under both chemical and biological reductive conditions, including bacterial and human azoreductases, which trigger the azo bond cleavage, leading to fluorescent 3-amino-BODIPY. We have also developed a practical enzymatic protocol, using an immobilized bacterial azoreductase that allows for the evaluation of these azo-based probes and can be used as a model for the less accessible and expensive human reductase NQO1. Quantum mechanical calculations uncover the restructuration of the topography of the S1 potential energy surface following the reduction of the azo moiety and rationalize the fluorescent quenching event through the mapping of an unprecedented pathway. Fluorescent microscopy experiments show that these azos can be used to visualize hypoxia-like conditions within living cells.
Droplet microfluidics enables the ultrahigh-throughput screening of the natural or man-made genetic diversity for industrial enzymes, with reduced reagent consumption and lower costs than ...conventional robotic alternatives. Here we describe an example of metagenomic screening for nucleoside 2'-deoxyribosyl transferases using FACS as a more widespread and accessible alternative than microfluidic on-chip sorters. This protocol can be easily adapted to directed evolution libraries by replacing the library construction steps and to other enzyme activities, e.g., oxidases, by replacing the proposed coupled assay.
Abstract Immobilization is a key enabling technology in applied biocatalysis that facilitates the separation, recovery, and reuse of heterogeneous biocatalysts. However, finding a consensus ...immobilization protocol for several enzymes forming a multi‐enzyme system is extremely difficult and relies on a combinatorial trial‐and‐error approach. Herein, we describe a protocol in which 17 different carriers functionalized with different reactive groups are tested in a 96‐well microtiter plate to screen up to 21 immobilization protocols for up to 18 enzymes. This screening includes an activity and stability assay to select the optimal immobilization chemistry to achieve the most active and stable heterogeneous biocatalysts. The information retrieved from the screening can be rationalized using a Python‐based application CapiPy. Finally, through scoring the screening results, we find the consensus immobilization protocol to assemble an immobilized four‐enzyme system to transform vinyl acetate into ( S )‐3‐hydroxybutyric acid. This methodology opens a path to speed up the prototyping of immobilized multi‐enzyme pathways for chemical manufacturing.
β-Hydroxy esters are essential building blocks utilised by the pharmaceutical and food industries in the synthesis of functional products. The asymmetric reduction of β-keto esters using cell-free ...enzymes presents a viable approach to manufacture enantiomerically pure β-hydroxy esters. However, the unbearable economic costs underlying enzymes and cofactors call for innovative approaches to maximise their reusability. Herein, we develop two self-sufficient Heterogeneous Biocatalysts (ssHBs) for the enantiodivergent reduction of β-keto esters to yield enantiomerically pure β-hydroxy esters. A thermophilic (
S
)-3-hydroxybutyryl-CoA dehydrogenase from
Thermus thermophilus
HB27 (TtHBDH) and an (
R
)-specific ketoreductase from
Lactobacillus kefir
(LkKRED) are selected, kinetically characterised, and immobilised onto macroporous agarose beads. Finally, the immobilised enzymes are coated with cationic polymers to co-immobilise the required redox cofactors. The resulting ssHBs catalyse the asymmetric reduction of β-keto esters without the exogenous supply of NAD(P)H and using 2-propanol as an ancillary electron donor. Then, we construct two enantiodivergent packed bed reactors (PBRs) integrating these two ssHBs and determine their optimal operational parameters through condition screening and kinetic simulations. The ssHBs in continuous flow operation exhibit good operational stability, illustrated by a maximum Space-Time Yield (STY) of 49.5 g L
−1
h
−1
for the continuous production of enantiopure ethyl 3-(
R
)-hydroxybutyrate over 21 days. Under these conditions, LkKRED and NADPH achieve total turnover numbers of 9.3 × 10
5
and 2.7 × 10
4
, respectively. Upon mass metric analysis, we conclude that these ssHBs meet the efficiency and sustainability standards to be implemented in some industrial processes, advancing the concept of self-sufficient biocatalysis for process intensification.
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.
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•The complex sucrose synthase from Nitrosomonas europaea was immobilized on a tailor-made glyoxyl agarose support.•The control of the multipoint covalent attachment was crucial to ...obtain optimal biocatalysts (activity and stability).•The thermal stability of the immobilized enzyme was mainly dependent on its orientation on the support.•The optimal immobilized biocatalyst was able to catalyze the synthesis of 210 mM of UDP-glucose for 3 reaction cycles.
Sucrose synthases (SuSys) can be used to synthesize cost-effective uridine 5′-diphosphate glucose (UDP-glc) or can be coupled to glycosyltransferases (GTs) for the continuous recycling of UDP-glc. In this study, we present the first report of the immobilization-stabilization of a SuSy by multipoint covalent attachment. This stabilization strategy is very complex for multimeric enzymes because a very intense multipoint attachment can promote a dramatic loss of activity and/or stability. The homotetrameric SuSy from Nitrosomonas europaea (SuSyNe) was immobilized on a glyoxyl agarose support through two different orientations. The first occurred at pH 8.5 through the surface area containing the greatest number of amino termini from several enzyme subunits. The second orientation occurred at pH 10 through the region of the whole enzyme containing the highest number of Lys residues. The multipoint covalent immobilization of SuSy on glyoxyl agarose at pH 10 provided a very significant stabilization factor under reaction conditions (almost 1000-fold more stable than soluble enzyme). Unfortunately, this important enzyme rigidification led to a dramatic loss of catalytic activity. A less stabilized conjugate, which was 65-fold more stable than the soluble form, preserved 64% of its initial catalytic activity. This derivative could be used for 3 reaction cycles and yielded approximately 210mM of UDP-glc per cycle. This optimal biocatalyst was modified with a polycationic polymer, polyethyleneimine (PEI), increasing its stability in the presence of the organic co-solvents necessary to glycosylate apolar antioxidants by GTs coupled to SuSy.
The in vitro synthesis of Coenzyme A (CoA)-thioester intermediates opens new avenues to transform simple molecules into more complex and multifunctional ones by assembling cell-free biosynthetic ...cascades. In this review, we have systematically cataloged known CoA-dependent enzyme reactions that have been successfully implemented in vitro. To faciliate their identification, we provide their UniProt ID when available. Based on this catalog, we have organized enzymes into three modules: activation, modification, and removal. i) The activation module includes enzymes capable of fusing CoA with organic molecules. ii) The modification module includes enzymes capable of catalyzing chemical modifications in the structure of acyl-CoA intermediates. And iii) the removal module includes enzymes able to remove the CoA and release an organic molecule different from the one activated in the upstream. Based on these reactions, we constructed a reaction network that summarizes the most relevant CoA-dependent biosynthetic pathways reported until today. From the information available in the articles, we have plotted the total turnover number of CoA as a function of the product titer, observing a positive correlation between both parameters. Therefore, the success of a CoA-dependent in vitro pathway depends on its ability to regenerate CoA, but also to regenerate other cofactors such as NAD(P)H and ATP.
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