A simple and direct ionic chromatography method to monitor galactose oxidase activity

Kaddouch, Eden; Cleveland, Maria E.; Navarro, David; Grisel, Sacha; Haon, Mireille; Brumer, Harry; Lafond, Mickaël; Berrin, Jean‐Guy; Bissaro, Bastien

doi:10.1039/d2ra04485d

Cited by 2 publications

(6 citation statements)

References 49 publications

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“…Finally, to assess the activity of both mutants on galactose‐containing oligosaccharides (e.g. d ‐Lac), a recently described method allowing the identification of oxidation products using HPAEC‐PAD technique was used [40]. For both Cgr AlcOx variants (M4F and M6), we confirmed the formation of a product corresponding to oxidized d ‐Lac (Fig.…”

Section: Resultssupporting

confidence: 61%

“…6A,B). According to previous NMR analysis on products resulting from Fgr GalOx‐WT [40], oxidation occurs at the C6 position of the galactose unit to the corresponding aldehyde, which is spontaneously hydrated to the gem ‐diol.…”

Section: Resultsmentioning

confidence: 99%

“…Samples for high‐performance anion‐exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD) analysis were prepared according to the protocol described by Kaddouch et al. [40]. Enzymatic reactions were performed with 70 n m of Fgr GalOx, or 2 μ m of Cgr AlcOx, Cgr AlcOx‐M4F or Cgr AlcOx‐M6 in sodium phosphate buffer (50 m m , pH 8.0), and using d ‐Lac (3 m m ) as substrate, 2 μ m of HRP and catalase and incubated in a Thermomixer (30 °C, 1000 r.p.m., up to 24 h).…”

Section: Methodsmentioning

confidence: 99%

“…6A,B). According to previous NMR analysis on products resulting from FgrGalOx-WT [40], oxidation occurs at the C6 position of the galactose unit to the corresponding aldehyde, which is spontaneously hydrated to the gem-diol. Overall, as observed in the first set of combined variants, and as expected, the accumulation of mutations seems proportional to the loss of activity mainly on nonactivated aryl and aliphatic alcohols and finally on activated substrates (Table 2B).…”

Section: Characterization Of a New Set Of Cgralcox Variantsmentioning

confidence: 96%

“…The reaction was stopped by heating to 100 °C (5 min) in a dry bath incubator. The mixture was then centrifuged (5 min, 12 000 9 g, 4 °C) and the supernatant was collected and diluted in Milli-Q water before injection into the HPAEC system following the analytic procedure described by Kaddouch et al [40].…”

Section: Abbreviationsmentioning

confidence: 99%

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

et al. 2023

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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.

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Section: Resultssupporting

confidence: 61%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Characterization Of a New Set Of Cgralcox Variantsmentioning

confidence: 96%

Section: Abbreviationsmentioning

confidence: 99%

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

et al. 2023

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Exploring the Intestinal Microbial Community of Lantang Pigs through Metagenome-Assembled Genomes and Carbohydrate Degradation Genes

Yang,

Fan,

Jin

et al. 2024

Fermentation

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High-fiber, low-cost agricultural byproducts offer a sustainable alternative for mitigating the competition for crops between humans and livestock. Pigs predominantly utilize dietary fibers through the process of microbial fermentation within the gut. This study explored the gut microbiota and the capacity for carbohydrate degradation in 30 individual Lantang pigs, a breed indigenous to China. Through metagenomic analysis, a total of 671 metagenome-assembled genomes (MAGs) were assembled and assigned into 14 bacterial and 1 archaeal phylum, including 97 species from uncultured microbes. The phylum with the highest abundance were identified as Bacillota_A, Bacteroidota, and Bacillota. Remarkably, the investigation revealed nearly 10,000 genes implicated in the degradation of carbohydrates, with a pronounced prevalence within five principal bacterial genera: Prevotella, Cryptobacteroides, Gemmiger, Vescimonas, and Faecousia. Additionally, 87 distinct types of carbohydrate-degrading enzymes were exclusively identified within the gut microbiota of the Lantang pig. These insights not only enhance our understanding of the microbial diversity specific to native Chinese pig breeds but also augment the body of research regarding porcine fiber degradation capabilities. The implications of this study are twofold: it provides strategic directions for optimizing feed efficiency and reducing breeding costs, and it furnishes an expanded gene pool for the microbial synthesis of industrial enzymes in the future.

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A simple and direct ionic chromatography method to monitor galactose oxidase activity

Abstract: Galactose oxidase (GalOx, EC.1.1.3.9) is one of the most extensively studied copper radical oxidases. Here, we show it can be monitored through the release of oxidized galactosylated products using a simple, direct and sensitive HPAEC-PAD method.

Cited by 2 publications

References 49 publications

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

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

Exploring the Intestinal Microbial Community of Lantang Pigs through Metagenome-Assembled Genomes and Carbohydrate Degradation Genes

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