Substrate Binding by the Catalytic Domain and Carbohydrate Binding Module of <i>Ruminococcus flavefaciens</i> FD-1 Xyloglucanase/Endoglucanase

Binding and thermodynamic properties of a carbohydrate binding module (CBM) and a glycoside hydrolase family 44 xyloglucanase/endoglucanase catalytic domain (CD) from <i>Ruminococcus flavefaciens</i>, both when separate and when linked to each other, have been quantified when binding various β-1,4-linked glucans and xylans. The three constructs bind cellotetraose, cellopentaose, and cellohexaose with association constants that increase with chain length. The CBM does not bind xylotetraose, xylopentaose, or xylohexaose. The CD appears to bind carboxymethylcellulose (CMC) and xylan only weakly, while the CBM and the CD/CBM bind them much more strongly than they bind the cellooligosaccharides. CMC is bound to a much greater degree than is xylan. Association constants for the cellooligosaccharides are in the order CBM ≪ CD < CD/CBM, while those on CMC and xylan are CD ≪ CBM ≪ CD/CBM. A synergistic effect was observed for the association constants of cellopentaose and cellohexaose with the CD/CBM when compared to the CD and CBM alone. Binding of all ligands by all three constructs is energetically favorable, enthalpy-driven, and subject to enthalpy–entropy compensation

Cell patterning and aggregate formation inside microwells.

<p><b>A)</b> Cell patterning. Cells were localized inside the microwells. <b>B)</b> After cell seeding, the cells in the microwell array were cultured in a petri dish and aggregates formed within 24 h. <b>C)</b> Once the aggregate formation is complete inside the microwells, they can be stained. <b>D)</b> Aggregates can be imaged inside microwells. <b>E)</b> Aggregates can be easily released from the microwells by gentle flushing with media for other applications.</p

Aggregate survival tests <i>in vitro</i>.

<p><b>A)</b> Subsets of microwell arrays with 2D monolayer of cell culture (2D) and aggregates of three sizes (S, M, and L). Hydrogen peroxide and anoxia/reoxygenation treatments were employed to induce cell death. EthD (red) and DAPI (blue) staining were performed for the determination of cell death. <b>B)</b> Quantification of dead CSP cells in 2D single layer culture and aggregates with variable diameters subjected to 200 µM-hydrogen peroxide treatment using EthD/DAPI fluorescent intensity ratio. Data were normalized to the vehicle groups of 2D monolayer culture and aggregates in three sizes. <b>C)</b> Quantification of dead CSP cells in 2D single layer culture and aggregates with variable diameters subjected to anoxia/reoxygenation using EthD/DAPI fluorescent intensity ratio. Data were normalized to the vehicle groups of 2D monolayer culture and aggregates in three sizes.</p