RESUMEN
Bacterial and fungal copper radical oxidases (CROs) from Auxiliary Activity Family 5 (AA5) are implicated in morphogenesis and pathogenesis. The unique catalytic properties of CROs also make these enzymes attractive biocatalysts for the transformation of small molecules and biopolymers. Despite a recent increase in the number of characterized AA5 members, especially from subfamily 2 (AA5_2), the catalytic diversity of the family as a whole remains underexplored. In the present study, phylogenetic analysis guided the selection of six AA5_2 members from diverse fungi for recombinant expression in Komagataella pfaffii (syn. Pichia pastoris) and biochemical characterization in vitro. Five of the targets displayed predominant galactose 6-oxidase activity (EC 1.1.3.9), and one was a broad-specificity aryl alcohol oxidase (EC 1.1.3.7) with maximum activity on the platform chemical 5-hydroxymethyl furfural (EC 1.1.3.47). Sequence alignment comparing previously characterized AA5_2 members to those from this study indicated various amino acid substitutions at active site positions implicated in the modulation of specificity.IMPORTANCEEnzyme discovery and characterization underpin advances in microbial biology and the application of biocatalysts in industrial processes. On one hand, oxidative processes are central to fungal saprotrophy and pathogenesis. On the other hand, controlled oxidation of small molecules and (bio)polymers valorizes these compounds and introduces versatile functional groups for further modification. The biochemical characterization of six new copper radical oxidases further illuminates the catalytic diversity of these enzymes, which will inform future biological studies and biotechnological applications.
Asunto(s)
Cobre , Oxidorreductasas , Filogenia , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Oxidorreductasas/química , Cobre/metabolismo , Saccharomycetales/genética , Saccharomycetales/enzimología , Especificidad por Sustrato , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Oxidorreductasas de Alcohol/química , Galactosa Oxidasa/genética , Galactosa Oxidasa/metabolismo , Galactosa Oxidasa/química , Alineación de Secuencia , Secuencia de Aminoácidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Dominio CatalíticoRESUMEN
Carbonyl cross-linkers are used to modify textiles and form resins, and are produced annually in megatonne volumes. Due to their toxicity toward the environment and human health, however, less harmful biobased alternatives are needed. This study introduces carbonyl groups to lactose and galactose using galactose oxidase from Fusarium graminearum (FgrGalOx) and pyranose dehydrogenase from Agaricus bisporus (AbPDH1) to produce four cross-linkers. Differential scanning calorimetry was used to compare cross-linker reactivity, most notably resulting in a 34 °C decrease in reaction peak temperature (72 °C) for FgrGalOx-oxidized galactose compared to unmodified galactose. Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), and proton nuclear magnetic resonance (1H NMR) spectroscopy were used to verify imine formation and amine and aldehyde depletion. Cross-linkers were shown to form gels when mixed with polyallylamine, with FgrGalOx-oxidized lactose forming gels more effectively than all other cross-linkers, including glutaraldehyde. Further development of carbohydrate cross-linker technologies could lead to their adoption in various applications, including in adhesives, resins, and textiles.
Asunto(s)
Reactivos de Enlaces Cruzados , Oxidación-Reducción , Poliaminas , Reactivos de Enlaces Cruzados/química , Poliaminas/química , Galactosa Oxidasa/química , Galactosa Oxidasa/metabolismo , Galactosa/química , Lactosa/química , Agaricus/química , Carbohidratos/químicaRESUMEN
Obtaining information about cellular interactions is fundamental to the elucidation of physiological and pathological processes. Proximity labeling technologies have been widely used to report cellular interactions in situ; however, the reliance on addition of tag molecules typically restricts their application to regions where tags can readily diffuse, while the application in, for example, solid tissues, is susceptible. Here, we propose an "in-situ-tag-generation mechanism" and develop the GalTag technology based on galactose oxidase (GAO) for recording cellular interactions within three-dimensional biological solid regions. GAO mounted on bait cells can in situ generate bio-orthogonal aldehyde tags as interaction reporters on prey cells. Using GalTag, we monitored the dynamics of cellular interactions and assessed the targeting ability of engineered cells. In particular, we recorded, for the first time, the footprints of Bacillus Calmette-Guérin (BCG) invasion into the bladder tissue of living mice, providing a valuable perspective to elucidate the anti-tumor mechanism of BCG.
Asunto(s)
Galactosa Oxidasa , Animales , Ratones , Galactosa Oxidasa/metabolismo , Galactosa Oxidasa/química , Humanos , Comunicación CelularRESUMEN
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.
Asunto(s)
Galactosa , Oro , Nanopartículas del Metal , Plata , Espectrometría Raman , Espectrometría Raman/métodos , Galactosa/química , Oro/química , Nanopartículas del Metal/química , Plata/química , Técnicas Biosensibles/métodos , Humanos , Peróxido de Hidrógeno/química , Límite de Detección , Galactosemias/diagnóstico , Galactosemias/sangre , Galactosa Oxidasa/química , Galactosa Oxidasa/metabolismo , Ácidos Borónicos/química , Compuestos de Sulfhidrilo/químicaRESUMEN
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.
Asunto(s)
Estabilidad de Enzimas , Galactosa Oxidasa , Presión Hidrostática , Galactosa Oxidasa/química , Galactosa Oxidasa/metabolismo , Calor , TemperaturaRESUMEN
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.
Asunto(s)
Cobre , Hidrógeno , Fenoles , Cobre/química , Galactosa Oxidasa/química , División del ADN , Metales , Piridinas , Ligandos , Cristalografía por Rayos XRESUMEN
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 3.25â mM (R2 =0.9985), a sensitivity of 122.78â µA mM-1 cm-2 , and a detection limit of 0.03â 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.
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Alcohol Bencilo , Galactosa Oxidasa , Galactosa Oxidasa/química , Oxidación-Reducción , Suministros de Energía Eléctrica , ElectrodosRESUMEN
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.
Asunto(s)
Cobre , Galactosa , Cobre/metabolismo , Galactosa/química , Oxidorreductasas/metabolismo , Galactosa Oxidasa/genética , Galactosa Oxidasa/química , Galactosa Oxidasa/metabolismo , Oxidación-Reducción , Ceruloplasmina , Alcoholes , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Cobre , Galactosa Oxidasa , Galactosa Oxidasa/química , Cobre/química , Oxidorreductasas de Alcohol , Oxidorreductasas/químicaRESUMEN
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 ËOH 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.
Asunto(s)
Galactosa Oxidasa , Estructuras Metalorgánicas , Aldehídos , Colorantes , Cobre , Galactosa , Galactosa Oxidasa/química , Galactosa Oxidasa/metabolismo , Peróxido de Hidrógeno/análisis , Peróxido de Hidrógeno/química , Estructuras Metalorgánicas/química , Oxidorreductasas , Oxígeno , Peroxidasa/metabolismo , Peroxidasas/químicaRESUMEN
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.
Asunto(s)
Galactosa Oxidasa , Vacunas Neumococicas , Polisacáridos Bacterianos , Streptococcus pneumoniae , Animales , Anticuerpos Antibacterianos/química , Anticuerpos Antibacterianos/inmunología , Galactosa Oxidasa/química , Galactosa Oxidasa/inmunología , Galactosa Oxidasa/metabolismo , Glicoconjugados , Ratones , Vacunas Neumococicas/química , Vacunas Neumococicas/inmunología , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/inmunología , Serogrupo , Streptococcus pneumoniae/química , Streptococcus pneumoniae/inmunología , Vacunas ConjugadasRESUMEN
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.
Asunto(s)
Furaldehído/análogos & derivados , Furaldehído/metabolismo , Galactosa Oxidasa/genética , Galactosa Oxidasa/metabolismo , Ingeniería de Proteínas , Biocatálisis , Catálisis , Dominio Catalítico , Furanos , Galactosa Oxidasa/química , Mutagénesis , Oxidación-ReducciónRESUMEN
Cu(I) active sites in metalloproteins are involved in O2 activation, but their O2 reactivity is difficult to study due to the Cu(I) d10 closed shell which precludes the use of conventional spectroscopic methods. Kß X-ray emission spectroscopy (XES) is a promising technique for investigating Cu(I) sites as it detects photons emitted by electronic transitions from occupied orbitals. Here, we demonstrate the utility of Kß XES in probing Cu(I) sites in model complexes and a metalloprotein. Using Cu(I)Cl, emission features from double-ionization (DI) states are identified using varying incident X-ray photon energies, and a reasonable method to correct the data to remove DI contributions is presented. Kß XES spectra of Cu(I) model complexes, having biologically relevant N/S ligands and different coordination numbers, are compared and analyzed, with the aid of density functional theory (DFT) calculations, to evaluate the sensitivity of the spectral features to the ligand environment. While the low-energy Kß2,5 emission feature reflects the ionization energy of ligand np valence orbitals, the high-energy Kß2,5 emission feature corresponds to transitions from molecular orbitals (MOs) having mainly Cu 3d character with the intensities determined by ligand-mediated d-p mixing. A Kß XES spectrum of the Cu(I) site in preprocessed galactose oxidase (GOpre) supports the 1Tyr/2His structural model that was determined by our previous X-ray absorption spectroscopy and DFT study. The high-energy Kß2,5 emission feature in the Cu(I)-GOpre data has information about the MO containing mostly Cu 3dx2-y2 character that is the frontier molecular orbital (FMO) for O2 activation, which shows the potential of Kß XES in probing the Cu(I) FMO associated with small-molecule activation in metalloproteins.
Asunto(s)
Cobre/metabolismo , Galactosa Oxidasa/metabolismo , Cobre/química , Teoría Funcional de la Densidad , Galactosa Oxidasa/química , Oxígeno/química , Oxígeno/metabolismo , Espectrometría por Rayos XRESUMEN
Galactose oxidase (GAO) contains a Cu(II)-ligand radical cofactor. The cofactor, which is autocatalytically generated through the oxidation of the copper, consists of a cysteine-tyrosine radical (Cys-Tyrâ¢) as a copper ligand. The formation of the cross-linked thioether bond is accompanied by a C-H bond scission on Tyr272 with few details known thus far. Here, we report the genetic incorporation of 3,5-dichlorotyrosine (Cl2-Tyr) and 3,5-difluorotyrosine (F2-Tyr) to replace Tyr272 in the GAOV previously optimized for expression through directed evolution. The proteins with an unnatural tyrosine residue are catalytically competent. We determined the high-resolution crystal structures of the GAOV, Cl2-Tyr272, and F2-Tyr272 incorporated variants at 1.48, 1.23, and 1.80 Å resolution, respectively. The structural data showed only one halogen remained in the cofactor, indicating that an oxidative carbon-chlorine/fluorine bond scission has occurred during the autocatalytic process of cofactor biogenesis. Using hydroxyurea as a radical scavenger, the spin-coupled hidden Cu(II) was observed by EPR spectroscopy. Thus, the structurally defined catalytic center with genetic unnatural tyrosine substitution is in the radical containing form as in the wild-type, i.e., Cu(II)-(Cl-Tyrâ¢-Cys) or Cu(II)-(F-Tyrâ¢-Cys). These findings illustrate a previously unobserved C-F/C-Cl bond cleavage in biology mediated by a mononuclear copper center.
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Carbono/química , Cobre/química , Flúor/química , Radicales Libres/química , Galactosa Oxidasa/metabolismo , Tirosina/química , Catálisis , Cristalografía por Rayos X , Evolución Molecular Dirigida , Espectroscopía de Resonancia por Spin del Electrón , Galactosa Oxidasa/química , Galactosa Oxidasa/genética , Cinética , Ligandos , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Estructura Terciaria de Proteína , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
Lipid rafts, highly ordered cell membrane domains mainly composed of cholesterol, sphingolipids, and protein receptors, serve as important functional platforms for regulation of lipid/protein interactions. The major predicament in lipid raft study is the lack of direct and robust visualization tools for in situ tracking raft components. To solve this issue, we herein report a proximity enzymatic glyco-remodeling strategy for direct and highly efficient lipid raft labeling and imaging on live cells. Through cofunctionalization of raft-specific recognition motif and glycan-remodeling enzyme on gold nanoparticles, the fabricated nanoprobe can be specifically guided to the raft domains to perform catalytic remodeling on neighboring glycans. Taking advantage of the abundant glycoconjugates enriched in lipid rafts, this elaborate design achieves the translation of one raft-recognition event to multiple raft-confined labeling operations, thus, significantly increasing the labeling efficiency and imaging sensitivity. The direct covalent labeling also enables in situ and long-term tracking of raft components in live cells. The method possesses broad applicability and potential expansibility, thus, will greatly facilitate the investigations on the complex composition, organization, and dynamics of lipid rafts.
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Toxina del Cólera/metabolismo , Galactosa Oxidasa/metabolismo , Lípidos/análisis , Polisacáridos/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Toxina del Cólera/química , Galactosa Oxidasa/química , Oro/química , Oro/metabolismo , Humanos , Nanopartículas del Metal/química , Polisacáridos/química , Células Tumorales CultivadasRESUMEN
Metalloenzymes catalyze many important reactions by managing the proton and electron flux at the enzyme active site. The motifs utilized to facilitate these transformations include hemilabile, redox-active, and so called proton responsive sites. Given the importance of incorporating and understanding these motifs in the area of coordination chemistry and catalysis, we highlight recent milestones in the field. Work incorporating the triad of hemilability, redox-activity, and proton responsivity into single ligand scaffolds will be described.
Asunto(s)
Dominio Catalítico , Galactosa Oxidasa/química , Galactosa Oxidasa/metabolismo , Protones , Ligandos , Modelos Moleculares , Oxidación-Reducción , Conformación ProteicaRESUMEN
For the construction of galactose biosensor, chitosan was electropolymerised onto the pencil graphite electrode. This chitosan modified pencil graphite electrode acts as good matrix for immobilization of enzyme nanoparticles of galactose oxidase. Development of this nanocomposite was further confirmed by Fourier transform infrared spectroscopy and scanning electron microscopy. The presence of chitosan makes the present galactose biosensor more efficient, reproducible and stable. The sensitivity was reported 7â¯×â¯10-3â¯mA/mM/cm2 with linear range from 0.05 to 25â¯mM and better detection limit of 0.05â¯mM. When the solution of galactose was spiked with 0.5â¯mM and 1â¯mM, the analytical recoveries were found 98.6% and 97.6%. A better storage stability was achieved (90days) when compared to earlier reported biosensors.
Asunto(s)
Técnicas Biosensibles/instrumentación , Galactosa Oxidasa/química , Galactosa/análisis , Quitosano/química , Electrodos , Enzimas Inmovilizadas/química , Grafito , Límite de Detección , Microscopía Electrónica de Rastreo , Nanocompuestos/química , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
A calix[4]arene ligand, in which two of the phenol functions are replaced by pyrazole units has been employed to mimic the His2 -Tyr2 (His: histidine, Tyr: tyrosine) ligand sphere within the active site of the galactose oxidase (GO). The calixarene backbone forces the corresponding copper(II) complex into a see-saw-type structure, which is hitherto unprecedented in GO modelling chemistry. It undergoes a one-electron oxidation that is centered at the phenolate donor leading to a copper-coordinated phenoxyl radical like in the GO. Accordingly, the complex was tested as a functional model and indeed proved capable of oxidizing benzyl alcohol to the respective aldehyde using two phenoxyl-radical equivalents as oxidants. Finally, the results show that the calixarene platform can be utilized to arrange donor functions to biomimetic binding pockets that allow for the creation of novel types of model compounds.
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Calixarenos/química , Galactosa Oxidasa/química , Oxígeno/química , Aldehídos/química , Secuencia de Aminoácidos , Catálisis , Dominio Catalítico , Complejos de Coordinación/química , Cobre/química , Técnicas Electroquímicas/métodos , Ligandos , Modelos Moleculares , Estructura Molecular , Níquel/química , Oxidación-Reducción , Fenoles/química , Unión Proteica , Zinc/químicaRESUMEN
Galactose oxidase catalyzes a two-electron oxidation, mainly from the C6 hydroxyl group of D-galactose, with the concomitant reduction of water to hydrogen peroxide. This enzyme is secreted by Fusarium species and has several biotechnological applications. In this study, a screening of galactose oxidase production among species of the Fusarium fujikuroi species complex demonstrated Fusarium subglutinans to be the main producer. The truncated F. subglutinans gaoA gene coding for the mature galactose oxidase was expressed from the prokaryotic vector pTrcHis2B in the E. coli Rosetta™ (DE3) strain. The purified recombinant enzyme presented temperature and pH optima of 30 °C and 7.0, respectively, KM of 132.6 ± 18.18 mM, Vmax of 3.2 ± 0.18 µmol of H2O2/min, kcat of 12,243 s-1, and a catalytic efficiency (kcat/KM) of 9.2 × 104 M-1 s-1. In the presence of 50% glycerol, the enzyme showed a T50 of 59.77 °C and was stable for several hours at pH 8.0 and 4 °C. Besides D-(+)-galactose, the purified enzyme also acted against D-(+)-raffinose, α-D-(+)-melibiose, and methyl-α-D-galactopyranoside, and was strongly inhibited by SDS. Although the F. subglutinans gaoA gene was successfully expressed in E. coli, its endogenous transcription was not confirmed by RT-PCR.
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Fusarium/enzimología , Galactosa Oxidasa/metabolismo , Galactosa/química , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fusarium/química , Galactosa/metabolismo , Galactosa Oxidasa/química , Galactosa Oxidasa/genética , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Concentración de Iones de Hidrógeno , Melibiosa/química , Melibiosa/metabolismo , Metilgalactósidos/química , Metilgalactósidos/metabolismo , Modelos Moleculares , Oxidación-Reducción , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Rafinosa/química , Rafinosa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , TemperaturaRESUMEN
The structures of three copper-containing complexes, namely (benzoato-κ2O,O')[(E)-2-({[2-(diethylamino)ethyl]imino}methyl)phenolato-κ3N,N',O]copper(II) dihydrate, [Cu(C7H5O2)(C13H19N2O)]·2H2O, 1, [(E)-2-({[2-(diethylamino)ethyl]imino}methyl)phenolato-κ3N,N',O](2-phenylacetato-κ2O,O')copper(II), [Cu(C8H7O2)(C13H19N2O)], 2, and bis[µ-(E)-2-({[3-(diethylamino)propyl]imino}methyl)phenolato]-κ4N,N',O:O;κ4O:N,N',O-(µ-2-methylbenzoato-κ2O:O')copper(II) perchlorate, [Cu2(C8H7O2)(C12H17N2O)2]ClO4, 3, have been reported and all have been tested for their activity in the oxidation of D-galactose. The results suggest that, unlike the enzyme galactose oxidase, due to the precipitation of Cu2O, this reaction is not catalytic as would have been expected. The structures of 1 and 2 are monomeric, while 3 consists of a dimeric cation and a perchlorate anion [which is disordered over two orientations, with occupancies of 0.64â (4) and 0.36â (4)]. In all three structures, the central Cu atom is five-coordinated in a distorted square-pyramidal arrangment (τ parameter of 0.0932 for 1, 0.0888 for 2, and 0.142 and 0.248 for the two Cu centers in 3). In each species, the environment about the Cu atom is such that the vacant sixth position is open, with very little steric crowding.