Name: Approcci di Biologia Strutturale e Chimica Analitica per la Caratterizzazione degli Enzimi Metabolici C-Glicosidici nel Microbiota Intestinale Umano (Video) | JoVE Journal
Uploaded: 2025-05-23T12:50:00.000+00:00
Duration: 13 min 35 s
Description: Scopri l'integrazione di biologia strutturale e chimica analitica per caratterizzare enzimi metabolici C-glicosidici nel microbiota intestinale umano. Questo metodo combina cristallografia a raggi X, LC-MS/MS e NMR.

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5 minutes

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<table><tbody><tr><td>2,6-dicloroindofenolo (DCPIP)</td><td>Solarbio Scienza e Tecnologia</td><td>D6350</td><td></td></tr><tr><td>2-Mercaptoetanolo (&beta;-ME)</td><td>Sigma-Aldrich</td><td>08168</td><td></td></tr><tr><td>&nbsp; 5 &volte; Carico di proteine colorante</td><td>Sangon Biotech</td><td>C508320</td><td></td></tr><tr><td>acido acetico</td><td>Shanghai MACKLIN</td><td>A111247</td><td></td></tr><tr><td>acetonitrile</td><td>Thermo Fisher Scientific&trade;? Invitrogen</td><td>A12147</td><td></td></tr><tr><td>Bruker Avance III HD 700 MHz NMR Spettrometro</td><td>Bruker</td><td>AVANCE III HD 700MHz</td><td></td></tr><tr><td>Bruker Ultrashield 400 Plus 400MHz</td><td>Bruker</td><td>AVANCE III 400 MHz</td><td></td></tr><tr><td>Centrifuga 5424R</td><td>Eppendorf</td><td>022620601</td><td></td></tr><tr><td>Chirascan più spettrometro CD</td><td>Fotofisica applicata</td><td>Chirascan &nbsp;</td><td></td></tr><tr><td>Daidzein</td><td>Shanghai Yuanye Bio-Technology</td><td>B20227</td><td></td></tr><tr><td>Daidzin</td><td>Shanghai Yuanye Bio-Technology</td><td>B20226</td><td></td></tr><tr><td>Dithiothreitol(DTT)</td><td>Solarbio Science and Technology</td><td>D8220</td><td></td></tr><tr><td>E. coli BL21 (DE3) cellule competenti</td><td>Thermo Fisher Scientific&trade;? Invitrogen</td><td>EC0114</td><td></td></tr><tr><td>Genisteina</td><td>Shanghai Yuanye Bio-Technology</td><td>S31565</td><td></td></tr><tr><td>Genistein-8-C-glucoside</td><td>Shanghai Yuanye Bio-Technology</td><td>BP2095</td><td></td></tr><tr><td>Genistin</td><td>Shanghai Yuanye Bio-Technology</td><td>S31591</td><td></td></tr><tr><td>Glucosio</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>GraphPad Prism 9</td><td>GraphPad Software</td><td>Versione? Prism 9</td><td>Calcola i parametri cinetici Km e kcat</td></tr><tr><td>Colonne SEC preparative HiLoad Superdex 200 pg</td><td>Cytiva</td><td>28989335</td><td></td></tr><tr><td>HisTrap HP His tag colonne per purificazione delle proteine</td><td>Cytiva</td><td>17524802</td><td></td></tr><tr><td>HiTrap Q FF colonna per cromatografia a scambio anionico</td><td>Cytiva</td><td>17515601</td><td></td></tr><tr><td>HKL-3000</td><td>HKL research Inc</td><td>&nbsp; versione 723</td><td></td></tr><tr><td>Colonna HPLC TC-C18(2), 170&Aring;, 5 &micro; m, 4,6 x 250 mm</td><td>Agilent Technologies</td><td>518925-902</td><td></td></tr><tr><td>Isopropil &beta;-D-1-tiogalattopiranoside? IPTG)</td><td>Sigma-Aldrich PHG0010</td><td></td><td></td></tr><tr><td>lattosio</td><td>Sigma-Aldrich</td><td>17814</td><td></td></tr><tr><td>LC-20AT, cromatografia liquida ad alte prestazioni</td><td>Shimadzu</td><td>LC20AT</td><td></td></tr><tr><td>Maltosio</td><td>Sigma-Aldrich</td><td>47288</td><td></td></tr><tr><td>Maltotriosio</td><td>Sigma-Aldrich</td><td>443713</td><td></td></tr><tr><td>Cloruro di manganese</td><td>Shanghai Yuanye Bio-Technology</td><td>S31112</td><td></td></tr><tr><td>Metanolo</td><td>Thermo Fisher Scientific&trade;? Invitrogen</td><td>M23142</td><td></td></tr><tr><td>NAD+</td><td>Solarbio Scienza e Tecnologia</td><td>N8110</td><td></td></tr><tr><td>Olympus SZX12 Stereomicroscopio</td><td>OLYMPUS&nbsp;</td><td>SZX7</td><td></td></tr><tr><td>fenilmetilsulfonilfluoruro (PMSF)</td><td>Sigma-Aldrich</td><td>P7626&nbsp;</td><td></td></tr><tr><td>Phenix</td><td>Il team di sviluppo di Phenix</td><td>1.21.2</td><td>Phenix è una suite software completa per la determinazione della struttura cristallina delle proteine, che incorpora i metodi SAD (Single-wavelength Anomalous Dispersion) e MR (Molecular Replacement) con il software Phaser.</td></tr><tr><td>Cromatografo liquido preparativo ad alte prestazioni</td><td>Waters</td><td>2545/2998</td><td></td></tr><tr><td>Kit di screening dei cristalli proteici</td><td>Hampton Research</td><td>HR2-110/HR2-112/HR2-144/HR2-126/HR2-098</td><td></td></tr><tr><td>Puerarin</td><td>Shanghai Yuanye Bio-Technology</td><td>B20446</td><td></td></tr><tr><td>Incubatore a scuotimento</td><td>Shanghai Zhichu Instrument Co., Ltd</td><td>&nbsp; ZQZY-AF8</td><td></td></tr><tr><td>Pelle di serpente&commercio; Membrana per dialisi, 10 kDa MWCO, 35 mm</td><td>Thermo Fisher Scientific&trade;? Invitrogen</td><td>88245</td><td></td></tr><tr><td>Amido solubile</td><td>Sigma-Aldrich</td><td>S9765</td><td></td></tr><tr><td>Sorvall LYNX 6000 Superspeed Centrifuga</td><td>Thermo Fisher Scientific&trade;</td><td></td><td></td></tr><tr><td>Lettore di micropiastre multimodale 75006590 SpectraMax i3x</td><td>DISPOSITIVI MOLECOLARI</td><td>SpectraMax i3x&nbsp;</td><td></td></tr><tr><td>Specifiche dello spettrofotometro per microvolumi SpectraMax QuickDrop DISPOSITIVI</td><td>MOLECOLARI</td><td>QuickDrop</td><td></td></tr><tr><td>Sucrosio</td><td>Sigma-Aldrich</td><td>S5016</td><td></td></tr><tr><td>SunFire C18 OBD Colonna di preparazione</td><td>Acque</td><td>186002741</td><td></td></tr><tr><td>Thermo Fisher Scientific Q EXACTIVE</td><td>Thermo Fisher Scientific&? Invitrogen</td><td>IQLAAEGAAPFALGMBDK</td><td></td></tr><tr><td>Ubiquitina-proteasi simile 1</td><td>Thermo Fisher Scientific&trade;? Invitrogen</td><td>12588018</td><td></td></tr><tr><td>Congelatore a bassissima temperatura</td><td>Thermo Fisher Scientific</td><td>905GP-ULTS</td><td></td></tr><tr><td>Concentratore centrifugo sottovuoto</td><td>Beijing JM Technology</td><td>CV600</td><td></td></tr><tr><td>Vitexin</td><td>Sigma-Aldrich</td><td>49513</td><td></td></tr></tbody></table>

approcci di biologia strutturale e chimica analitica per la caratterizzazione degli enzimi metabolici c-glicosidici nel microbiota intestinale umano

Structural Biology and Analytical Chemistry Approaches for Characterizing C-Glycoside Metabolic Enzymes in Human Gut Microbiota

Seguendo le procedure descritte in Figura 1, abbiamo prima espresso i geni DgpA, DgpB e DgpC in E. coli Celle BL21(DE3). Poiché DgpB e DgpC formano un complesso enzimatico stabile (complesso DgpB/C)10,26,27,34, le cellule che esprimono queste due proteine sono state combinate per la co-purificazione. Dopo la lisi cellulare,...

Approcci di Biologia Strutturale e Chimica Analitica per la Caratterizzazione degli Enzimi Metabolici C-Glicosidici nel Microbiota Intestinale Umano

The gut microbiota has emerged as a key modulator of host health, particularly because of its capacity to metabolize dietary glycosides. While ...

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Un metodo specifico e sensibile al fine di conoscere il profilo di espressione di antigeni glicosfingolipidi in organi e cellule immunitarie è descritto. Il metodo sfrutta la spettrometria di massa a trappola ionica consentendo graduale frammentazione delle molecole glicosfingolipidi per l'analisi strutturale rispetto agli standard sintetizzati chimicamente.

Spettrometria di massa, di antigeni glicosfingolipide

Fonte: Hu, Y. et.al., Analisi delle Interazioni tra Endobiotici e Microbiota Intestinale Umano Utilizzando Sistemi di Fermentazione in Vitro Bath. J. Vis. Esp. (2019)Questo video dimostra l'analisi della produzione di acidi grassi a catena corta da parte del microbiota intestinale umano in risposta agli esosaccaridi. Questo evidenzia la separazione e quantificazione dei prodotti della fermentazione microbica tramite cromatografia gassosa. I risultati rivelano che gli esosaccaridi modulano i...

Indagine dell'attività microbica intestinale umana sugli esosaccacaridi

La comunità batterica attivo associato con l'intestino di Spodoptera littoralis, è stato determinato mediante stabile-isotopo probing (SIP) accoppiato a pyrosequencing. Utilizzando questa metodologia, l'identificazione delle specie di batteri metabolicamente attivi all'interno della comunità è stato fatto con alta risoluzione e precisione.

Identificazione dei batteri metabolicamente attivo nell&#39;intestino del generalista<em&gt; Spodoptera littoralis</em&gt; Via DNA isotopi stabili Probing Utilizzo<sup&gt; 13</sup&gt; C-glucosio

Viene descritto un test ad alto rendimento per lo screening enzimatico. Questo kit di saggi multiplexato e pronto all'uso comprende substrati prescelti diC hromogenic Polymer Hydrogel (CPH) e substrati di C insolubileC iomogenico Biomass (ICB). Gli enzimi bersaglio sono gli endoenzimi che degradano i polisaccaridi e le proteasi.

High-throughput screening degli enzimi carboidrati di degradazione Utilizzando Novel insolubile Chromogenic Substrate Assay Kit

Qui, presentiamo un protocollo per la coltura del microbiota dell'intestino, del colon in vitro, , utilizzando una serie di bioreattori che simulano le condizioni fisiologiche del tratto gastro intestinale.

L'applicazione di avanzate In Vitro coltura tecnologia per studiare il Microbiota dell'intestino umano

Descriviamo qui il rilevamento ad alta velocità e la quantificazione degli oligosaccaridi del latte umano fucosylated (HMOs) utilizzando un biosensore di cellule intere. Inoltre dimostriamo qui, l'adattamento di questa piattaforma nei confronti di ceppi di produzione HMO, concentrandosi sul miglioramento del rapporto segnale-rumore.

Analisi di Fucosylated Human latte trisaccaridi nel contesto biotecnologica utilizzando geneticamente codificato biosensori

Descritto qui è un protocollo per studiare le interazioni tra endobiotici e microbiota intestinale umano utilizzando sistemi di fermentazione in vitro.

Analisi delle interazioni tra endobiotici e microbiota intestinale umana utilizzando sistemi di fermentazione bagno in vitro

Di seguito è descritta una procedura operativa standard semplificata per la profilazione del microbioma che utilizza il sequenziamento e l'analisi metagenomica del rRNA 16S utilizzando strumenti liberamente disponibili. Questo protocollo aiuterà i ricercatori che non hanno familiarità con il campo del microbioma e quelli che richiedono aggiornamenti sui metodi per ottenere una profilazione batterica a una risoluzione più elevata.

Analisi microbiota utilizzando PCR in due fasi e sequenziamento genico rRNA 16S di nuova generazione

La ricerca aperta consente l'identificazione di glicopeptidi decorati con composizioni di glicani precedentemente sconosciute. All'interno di questo articolo, viene presentato un approccio semplificato per intraprendere la ricerca aperta e le successive ricerche di glicopeptidi focalizzate sul glicano per campioni batterici utilizzando Acinetobacter baumannii come modello.

L'applicazione di approcci aperti basati sulla ricerca per l'identificazione di glicopeptidi legati all'O di Acinetobacter baumannii

Questo studio stabilisce una serie di procedure operative standard (SOP) per lo screening e l'isolamento efficiente dei batteri intestinali in grado di scindere i glicosidi C.

Screening e isolamento dei batteri intestinali che scindono il glicoside C

Scopri l'integrazione di biologia strutturale e chimica analitica per caratterizzare enzimi metabolici C-glicosidici nel microbiota intestinale umano. Questo metodo combina cristallografia a raggi X, LC-MS/MS e NMR.

Approcci di Biologia Strutturale e Chimica Analitica per la Caratterizzazione degli Enzimi Metabolici C-Glicosidici nel Microbiota Intestinale Umano (Video) | JoVE Journal

Approcci di Biologia Strutturale e Chimica Analitica per la Caratterizzazione degli Enzimi Metabolici C-Glicosidici nel Microbiota Intestinale Umano

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