Simple and sensitive galactose monitoring based on capillary SERS sensor.

Galactosemia, a severe genetic metabolic disorder, results from the absence of galactose-degrading enzymes, leading to harmful galactose accumulation. In this study, we introduce a novel capillary-based surface-enhanced Raman spectroscopy (SERS) sensor for convenient and sensitive galactose detection. The developed sensor enhances SERS signals by introducing gold nanoparticles (Au NPs) onto the surface of silver nanoshells (Ag NSs) within a capillary, creating Ag NSs with Au NPs as satellites. Utilizing 4-mercaptophenylboronic acid (4-MPBA) as a Raman reporter molecule, the detection method relies on the conversion of 4-MPBA to 4-mercaptophenol (4-MPhOH) driven by hydrogen peroxide (H2O2) generated during galactose oxidation by galactose oxidase (GOx). A new SERS signal was observed, which was generated by H2O2 produced when galactose and GOx reacted. Our strategy yielded a quantitative change in the SERS signal, specifically in the band intensity ratio of 998 to 1076 cm-1 (I998/I1076) as the galactose concentration increased. Our capillary-based SERS biosensor provides a promising platform for early galactosemia diagnosis.

Stabilization of galactose oxidase by high hydrostatic pressure: Insights on the role of cavities size.

High hydrostatic pressure stabilized galactose oxidase (GaOx) at 70.0-80.0°C against thermal inactivation. The pseudo-first-order rate constant of inactivation kinact decreased by a factor of 8 at 80°C and by a factor of 44 at 72.5°C. The most pronounced effect of pressure was at the lowest studied temperature of 70.0°C with an activation volume of inactivation ΔV‡ of 78.8 cm3 mol-1. The optimal pressure against thermal inactivation was between 200 and 300 MPa. Unlike other enzymes, as temperature increased the ΔV‡ of inactivation decreased, and as pressure increased the activation energy of inactivation Eai increased. Combining the results for GaOx with earlier research on the pressure-induced stabilization of other enzymes suggests that ΔV‡ of inactivation correlates with the total molar volume of cavities larger than ~100 Å3 in enzyme monomers for enzymes near the optimal pH and whose thermal unfolding is not accompanied by oligomer dissociation.

Effect of Protection Polymer Coatings on the Performance of an Amperometric Galactose Biosensor in Human Plasma.

Galactose monitoring in individuals allows the prevention of harsh health conditions related to hereditary metabolic diseases like galactosemia. Current methods of galactose detection need development to obtain cheaper, more reliable, and more specific sensors. Enzyme-containing amperometric sensors based on galactose oxidase activity are a promising approach, which can be enhanced by means of their inclusion in a redox polymer coating. This strategy simultaneously allows the immobilization of the biocatalyst to the electroactive surface and hosts the electron shuttling units. An additional deposition of capping polymers prevents external interferences like ascorbic or uric acid as well as biofouling when measuring in physiological fuels. This work studies the protection effect of poly(2-methacryloyloxyethyl phosphorylcholine-co-glycidyl methacrylate (MPC) and polyvinylimidazole-polysulfostyrene (P(VI-SS)) when incorporated in the biosensor design for the detection of galactose in human plasma.

Crystal structure of a phenoxyl radical complex relevant to the metal site of the galactose oxidase enzyme: A facile one-pot synthesis, evidence for hydrogen atom transfer and DNA cleavage via self-activation.

Copper complexes [Cu(L1H)ClO4] (1) and [Cu(L2)NO3] (2), which are relevant to the metal site of the galactose oxidase enzyme, were synthesized and characterized by different spectroscopic methods. L1H2 and L2H2 [where L1H2 stands for 2,2'-((1E,1'E)(2,2'-(pyridine-2,6-diyl)bis(2-phenylhydrazin-2-yl-1-ylidene))bis(methanylylidene))diphenol and L2H2 stands for 6,6'-((1E,1'E)-(2,2'-(pyridine-2,6-diyl)bis(2-phenylhydrazin-2-yl-1-ylidene))bis(methanylylidene))bis(2,4-di-tert-butylphenol), H stands for dissociable proton] are pentadentate ligands. These ligands provide pyridyl N, two imine N, and two non-innocent phenoxyl and phenolato O donors, forming complex 1 as a non-radical complex, while complex 2 is a phenoxyl radical complex. The molecular structures of complexes 1 and 2 were authenticated by X-ray crystallography. Benzyl alcohol oxidation was investigated, and the conversion of 9,10-dihydroanthracene to anthracene was examined to scrutinize the H-atom abstraction reaction. Nuclease activity with complexes 1 and 2 was investigated by self-activated plasmid DNA (pBR322) cleavage. Non-innocent properties of the ligand-containing phenolato function were investigated by DFT calculations.

Overcoming Biochemical Limitations of Galactose Oxidase through the Design of a Solid-Supported Self-Sufficient Biocatalyst.

Galactose Oxidase (GalOx) has gained significant interest in biocatalysis due to its ability for selective oxidation beyond the natural oxidation of galactose, enabling the production of valuable derivatives. However, the practical application of GalOx has been hindered by the limited availability of active and stable biocatalysts, as well as the inherent biochemical limitations such as oxygen (O2 ) dependency and the need for activation. In this study, we addressed these challenges by immobilizing GalOx into agarose-based and Purolite supports to enhance its activity and stability. Additionally, we identified and quantified the oxygen supply limitation into solid catalysts by intraparticle oxygen sensing showing a trade-off between the amount of protein loaded onto the solid support and the catalytic effectiveness of the immobilized enzyme. Furthermore, we coimmobilized a heme-containing protein along with the enzyme to function as an activator. To evaluate the practical application of the immobilized GalOx, we conducted the oxidation of galactose in an instrumented aerated reactor. The results showcased the efficient performance of the immobilized enzyme in the 8 h reaction cycle. Notably, the GalOx immobilized into dextran sulfate-activated agarose exhibited improved stability, overcoming the need for a soluble activator supply, and demonstrated exceptional performance in galactose oxidation. These findings offer promising prospects for the utilization of GalOx in technical biocatalytic applications.

Co-immobilization of galactose oxidase, catalase, and Mn-superoxide dismutase for efficient conversion of 5-hydroxymethylfurfural to 2,5-diformylfuran in water.

The three enzymes galactose oxidase (GO), catalase (CAT), and Mn-superoxide dismutase (SOD) were simultaneously immobilized by coordinating to CuII in phosphate buffer saline. The biocatalyst GO&CAT&SOD@CuII was used for the conversion of 5-hydroxymethylfurfural (HMF). The immobilized GO catalyzes the oxidation of HMF to 2,5-diformylfuran (DFF), concomitantly the co-substrate O2 is reduced to hydrogen peroxide (H2O2). A portion of the byproduct H2O2 is broken down to O2 and H2O by the co-immobilized CAT, and the evolved O2 can be recycled and used as the co-substrate. A portion of the byproduct H2O2 is broken down to produce hydroxyl radicals •OH under the synergistic catalysis of the immobilized SOD and coordinated CuII, and the produced •OH can reactivate the immobilized galactose oxidase. Two aspects contribute to the high catalytic efficiency by GO&CAT&SOD@CuII: the reactivation of the immobilized galactose oxidase by producing •OH and the enrichment of the co-substate O2 by recycling the produced O2. For the conversion of 10 mM HMF, GO&CAT&SOD@CuII (with encapsulated GO 0.2 mg/mL) achieved 97% HMF conversion within 2 h reaction. In contrast, free galactose oxidase M3-5 variant (ACS Catalysis 2018, 8, 4025) (0.2 mg/mL) achieved 25.3% HMF conversion within 2 h reaction. All the reactions were carried out in pure water, not in PBS.

A Flow Cytometry-Based Ultrahigh-Throughput Screening Method for Directed Evolution of Oxidases.

Oxidases are of interest to chemical and pharmaceutical industries because they catalyze highly selective oxidations. However, oxidases found in nature often need to be re-engineered for synthetic applications. Herein, we developed a versatile and robust flow cytometry-based screening platform "FlOxi" for directed oxidase evolution. FlOxi utilizes hydrogen peroxide produced by oxidases expressed in E. coli to oxidize Fe2+ to Fe3+ (Fenton reaction). Fe3+ mediates the immobilization of a His6 -tagged eGFP (eGFPHis ) on the E. coli cell surface, ensuring the identification of beneficial oxidase variants by flow cytometry. FlOxi was validated with two oxidases-a galactose oxidase (GalOx) and a D-amino acid oxidase (D-AAO)-yielding a GalOx variant (T521A) with a 4.4-fold lower Km value and a D-AAO variant (L86M/G14/A48/T205) with a 4.2-fold higher kcat than their wildtypes. Thus, FlOxi can be used for the evolution of hydrogen peroxide-producing oxidases and applied for non-fluorescent substrates.

Tailoring Galactose Oxidase for Self-Powered Benzyl Alcohol Sensing.

Benzyl alcohol (BnOH) is a widely-used preservative in a variety of cosmetics, but the excess addition (≥1.0 %) may cause strong symptoms such as nausea, gastrointestinal irritation, convulsion, even death, making it crucial to monitor and control the addition quantity. Herein, we have developed a test-strip-like BnOH detection method via tailoring a galactose oxidase (GOase) towards BnOH oxidation and preparing a self-powered electrochromic strip for BnOH concentration visualization. A double-substituted GOase variant (Y329S/R330F), on the basis of the reported GOase M1 , has been obtained by semi-rational design with a 24.6-fold improved activity towards BnOH compared to GOase M1 . The GOase Y329S/R330F electrode has a response to BnOH with a linear range of 0.04 to 3.25 mM (R2 =0.9985), a sensitivity of 122.78 µA mM-1 cm-2 , and a detection limit of 0.03 mM (S/N=3). Coupling an electrochromic Prussian blue (PB) cathode helps the successful sensing visualization without any further power supply. The present sensing is more convenient and user-friendly than the generally used gas chromatography (GC) and high performance liquid chromatography (HPLC), and brings a more accessible solution to the field of quality controlling.

Rational engineering of AA5_2 copper radical oxidases to probe the molecular determinants governing their substrate selectivity.

Fungal copper radical oxidases (CROs) from the Auxiliary Activity family 5 (AA5) constitute a group of metalloenzymes that oxidize a wide panel of natural compounds, such as galactose-containing saccharides or primary alcohols, into product derivatives exhibiting promising biotechnological interests. Despite a well-conserved first copper-coordination sphere and overall fold, some members of the AA5_2 subfamily are incapable of oxidizing galactose and galactosides but conversely efficiently catalyse the oxidation of diverse aliphatic alcohols. The objective of this study was to understand which residues dictate the substrate preferences between alcohol oxidases and galactose oxidases within the AA5_2 subfamily. Based on structural differences and molecular modelling predictions between the alcohol oxidase from Colletotrichum graminicola (CgrAlcOx) and the archetypal galactose oxidase from Fusarium graminearum (FgrGalOx), a rational mutagenesis approach was developed to target regions or residues potentially driving the substrate specificity of these enzymes. A set of 21 single and multiple CgrAlcOx variants was produced and characterized leading to the identification of six residues (W39, F138, M173, F174, T246, L302), in the vicinity of the active site, crucial for substrate recognition. Two multiple CgrAlcOx variants, i.e. M4F (W39F, F138W, M173R and T246Q) and M6 (W39F, F138W, M173R, F174Y, T246Q and L302P), exhibited a similar affinity for carbohydrate substrates when compared to FgrGalOx. In conclusion, using a rational site-directed mutagenesis approach, we identified key residues involved in the substrate selectivity of AA5_2 enzymes towards galactose-containing saccharides.

Copper radical oxidases: galactose oxidase, glyoxal oxidase, and beyond!

The copper radical oxidases (CROs) are an evolutionary and functionally diverse group of enzymes established by the historically significant galactose 6-oxidase and glyoxal oxidase from fungi. Inducted in 2013, CROs now constitute Auxiliary Activity Family 5 (AA5) in the Carbohydrate-Active Enzymes (CAZy) classification. CROs catalyse the two-electron oxidation of their substrates using oxygen as the final electron acceptor and are particularly distinguished by a cross-linked tyrosine-cysteine co-factor that is integral to radical stabilization. Recently, there has been a significant increase in the biochemically and structurally characterized CROs, which has revealed an expanded natural diversity of catalytic activities in the family. This review provides a brief historical introduction to CRO biochemistry and structural biology as a foundation for an update on current advances in CRO enzymology, biotechnology, and biology across kingdoms of life.

Preparation of iminosugars from aminopolyols via selective oxidation using galactose oxidase.

Minimally protected aminopolyols are novel substrates for the galactose oxidase variant F2. Site-selective oxidation proceeds at the terminal primary alcohol, followed by spontaneous cyclisation to afford stable hemiaminal/hemiacetal anomers of the piperidine and azepane scaffolds, with isolated yields of up to 94%. Simultaneous deprotection and reduction occured readily to afford valuable and biologically relevant iminosugars.

Hemilabile Amine-Functionalized Efficient Azo-Aromatic Cu-Catalysts Inspired by Galactose Oxidase: Impact of Amine Sidearm on Catalytic Aerobic Oxidation of Alcohols.

A series of azo-aromatic copper(II) complexes, [1a-g] and a Cu(I) complex, [1h], with varying amine-functionalized hemilabile pincer-like [HL1-3] and [L1,2], methyl-substituted azo [L3], and imine [L4] ligands, were synthesized and characterized. These complexes were investigated for aerobic oxidation of a variety of aromatic alcohols in the presence of 2.0 mol % precatalysts [1a-g], cobaltocene (2.0 mol %), N-methyl imidazole (NMI) (8.0 mol %), and TEMPOH (2.0 mol %) at room temperature. The Cu(I) complex (1h) acted as a catalyst in the absence of cobaltocene. To understand the mechanism, detailed experimental and theoretical studies have been performed with the representative complex [1a], which has suggested a new kind of mechanism involving a Cu(II)/Cu(I) redox couple. Cobaltocene acts as a reductant to [1a] to generate a Cu(I) complex, which activates dioxygen in the presence of NMI. TEMPOH transfers a hydrogen atom to the activated dioxygen with the generation of TEMPO•, which further participates in α-C-H bond activation in the Cu(II)-alkoxide intermediate in an intermolecular fashion in the catalytic cycle. The amine sidearm in the ligand backbone of the complexes has a significant role in catalytic activity. Complexes with amine sidearms are more effective than complexes without them. Moreover, the aliphatic secondary amine sidearm is more efficient among the amine sidearm than the aromatic secondary amine and tertiary amines. The amine sidearm that remained coordinated to the Cu(II) center is hemilabile, and it facilitates alcohol coordination in the catalytic process. Alcohol coordination was the rate-limiting step, and it was supported by the isotope effect study on benzyl alcohol, substitution effect on the amine moiety of the ligands, and DFT calculation. The hemilabile amine sidearm of the coordinated ligand also acted as a base in deprotonating the alcoholic O-H proton and acted as an acid in releasing H2O2 during the catalysis.

One step cascade detection of galactose based on a galactose oxidase-composited peroxidase nanozyme.

Abnormal galactose metabolism is the main cause of galactosemia, which makes the accurate and rapid analysis of galactose levels in food and organism the key issue at present. In this study, a novel strategy for one-step galactose determination was proposed based on galactose oxidase and copper-based metal-organic framework complexes (GAOx@MOF) with dual catalytic activities at neutral pH. Typically, GAOx catalyzes the oxidation of the C6 hydroxyl group of D-galactose to generate an aldehyde (D-galactose-hexanedial), and coupled with the reduction of dioxygen to H2O2, which was immediately transformed to ˙OH by mimicking peroxidase activity and at the same time oxidized ABTS to a green product with a clear colorimetric signal. The whole process was completed using one buffer, which simplified the procedure and increased the sensitivity. Moreover, the proposed method can also be used for the quantitative analysis of galactose. It showed a good linear relationship at 20-1000 µM, while the LOD was 6.67 µM. Furthermore, the strategy has been successfully utilized for galactose determination in milk samples, which proved its promising applications in clinical analysis and the food industry.

Formation and Reactivity of a Fleeting NiIII Bisphenoxyl Diradical Species.

Cytochrome P450s and Galactose Oxidases exploit redox active ligands to form reactive high valent intermediates for oxidation reactions. This strategy works well for the late 3d metals where accessing high valent states is rather challenging. Herein, we report the oxidation of NiII (salen) (salen=N,N'-bis(3,5-di-tert-butyl-salicylidene)-1,2-cyclohexane-(1R,2R)-diamine) with mCPBA (meta-chloroperoxybenzoic acid) to form a fleeting NiIII bisphenoxyl diradical species, in CH3 CN and CH2 Cl2 at -40 °C. Electrochemical and spectroscopic analyses using UV/Vis, EPR, and resonance Raman spectroscopies revealed oxidation events both on the ligand and the metal centre to yield a NiIII bisphenoxyl diradical species. DFT calculations found the electronic structure of the ligand and the d-configuration of the metal center to be consistent with a NiIII bisphenoxyl diradical species. This three electron oxidized species can perform hydrogen atom abstraction and oxygen atom transfer reactions.

On the Mediated Electron Transfer of Immobilized Galactose Oxidase for Biotechnological Applications.

Invited for the cover of this issue are Felipe Conzuelo, Wolfgang Schuhmann, and co-workers at the Ruhr University Bochum. The image depicts the electrochemical conversion of glycerol and 5-(hydroxymethyl)furfural with an electrode made up of galactose oxidase electrically wired with a redox polymer. Read the full text of the article at 10.1002/chem.202200868.

Enzymatic Cascade for the Synthesis of 2,5-Furandicarboxylic Acid in Biphasic and Microaqueous Conditions: 'Media-Agnostic' Biocatalysts for Biorefineries.

5-hydroxymethylfurfural (HMF) is produced upon dehydration of C6 sugars in biorefineries. As the product, it remains either in aqueous solutions, or is in situ extracted to an organic medium (biphasic system). For the subsequent oxidation of HMF to 2,5-furandicarboxylic acid (FDCA), 'media-agnostic' catalysts that can be efficiently used in different conditions, from aqueous to biphasic, and to organic (microaqueous) media, are of interest. Here, the concept of a one-pot biocatalytic cascade for production of FDCA from HMF was reported, using galactose oxidase (GalOx) for the formation of 2,5-diformylfuran (DFF), followed by the lipase-mediated peracid oxidation of DFF to FDCA. GalOx maintained its catalytic activity upon exposure to a range of organic solvents with only 1 % (v/v) of water. The oxidation of HMF to 2,5-diformylfuran (DFF) was successfully established in ethyl acetate-based biphasic or microaqueous systems. To validate the concept, the reaction was conducted at 5 % (v/v) water, and integrated in a cascade where DFF was subsequently oxidized to FDCA in a reaction catalyzed by Candida antarctica lipase B.

On the Mediated Electron Transfer of Immobilized Galactose Oxidase for Biotechnological Applications.

The use of enzymes as catalysts in chemical synthesis offers advantages in terms of clean and highly selective transformations. Galactose oxidase (GalOx) is a remarkable enzyme with several applications in industrial conversions as it catalyzes the oxidation of primary alcohols. We have investigated the wiring of GalOx with a redox polymer; this enables mediated electron transfer with the electrode surface for its potential application in biotechnological conversions. As a result of electrochemical regeneration of the catalytic center, the formation of harmful H2 O2 is minimized during enzymatic catalysis. The introduced bioelectrode was applied to the conversion of bio-renewable platform materials, with glycerol as model substrate. The biocatalytic transformations of glycerol and 5-hydroxymethylfurfural (HMF) were investigated in a circular flow-through setup to assess the possibility of substrate over-oxidation, which is observed for glycerol oxidation but not during HMF conversion.

Harnessing galactose oxidase in the development of a chemoenzymatic platform for glycoconjugate vaccine design.

In the preparation of commercial conjugate vaccines, capsular polysaccharides (CPSs) must undergo chemical modification to generate the reactive groups necessary for covalent attachment to a protein carrier. One of the most common approaches employed for this derivatization is sodium periodate (NaIO4) oxidation of vicinal diols found within CPS structures. This procedure is largely random and structurally damaging, potentially resulting in significant changes in the CPS structure and therefore its antigenicity. Additionally, periodate activation of CPS often gives rise to heterogeneous conjugate vaccine products with variable efficacy. Here, we explore the use of an alternative agent, galactose oxidase (GOase) isolated from Fusarium sp. in a chemoenzymatic approach to generate a conjugate vaccine against Streptococcus pneumoniae. Using a colorimetric assay and NMR spectroscopy, we found that GOase generated aldehyde motifs on the CPS of S. pneumoniae serotype 14 (Pn14p) in a site-specific and reversible fashion. Direct comparison of Pn14p derivatized by either GOase or NaIO4 illustrates the functionally deleterious role chemical oxidation can have on CPS structures. Immunization with the conjugate synthesized using GOase provided a markedly improved humoral response over the traditional periodate-oxidized group. Further, functional protection was validated in vitro by measure of opsonophagocytic killing and in vivo through a lethality challenge in mice. Overall, this work introduces a strategy for glycoconjugate development that overcomes limitations previously known to play a role in the current approach of vaccine design.

Galactose Oxidase Enables Modular Assembly of Conjugates from Native Antibodies with High Drug-to-Antibody Ratios.

The potential of antibody conjugates with high drug loading in anticancer therapy has recently been highlighted by the approval of Trastuzumab deruxtecan and Sacituzumab govitecan. These biopharmaceutical approaches have spurred interest in bioconjugation strategies with high and defined degrees of drug-to-antibody ratio (DAR), in particular on native antibodies. Here, a glycoengineering methodology was developed to generate antibody drug conjugates with DAR of up to eight, by combining highly selective enzymatic galactosylation and oxidation with biorthogonal tandem Knoevenagel-Michael addition chemistry. This four-step approach offers a selective route to conjugates from native antibodies with high drug loading, and thus illustrates how biocatalysis can be used for the generation of biopharmaceuticals using mild reaction conditions.

Toward scalable biocatalytic conversion of 5-hydroxymethylfurfural by galactose oxidase using coordinated reaction and enzyme engineering.

5-Hydroxymethylfurfural (HMF) has emerged as a crucial bio-based chemical building block in the drive towards developing materials from renewable resources, due to its direct preparation from sugars and its readily diversifiable scaffold. A key obstacle in transitioning to bio-based plastic production lies in meeting the necessary industrial production efficiency, particularly in the cost-effective conversion of HMF to valuable intermediates. Toward addressing the challenge of developing scalable technology for oxidizing crude HMF to more valuable chemicals, here we report coordinated reaction and enzyme engineering to provide a galactose oxidase (GOase) variant with remarkably high activity toward HMF, improved O2 binding and excellent productivity (>1,000,000 TTN). The biocatalyst and reaction conditions presented here for GOase catalysed selective oxidation of HMF to 2,5-diformylfuran offers a productive blueprint for further development, giving hope for the creation of a biocatalytic route to scalable production of furan-based chemical building blocks from sustainable feedstocks.