Coimmobilization of β‑Agarase and α‑Neoagarobiose Hydrolase for Enhancing the Production of 3,6-Anhydro‑l‑galactose

Here we report a simple and efficient method to produce 3,6-anhydro-l-galactose (l-AHG) and agarotriose (AO3) in one step by a multienzyme system with the coimmobilized β-agarase AgWH50B and α-neoagarobiose hydrolase K134D. K134D was obtained by AgaWH117 mutagenesis and showed improved thermal stability when immobilized via covalent bonds on functionalized magnetic nanoparticles. The obtained multienzyme biocatalyst was characterized by Fourier transform infrared spectroscopy (FTIR). Compared with free agarases, the coimmobilized agarases exhibited a relatively higher agarose-to-l-AHG conversion efficiency. The yield of l-AHG obtained with the coimmobilized agarases was 40.6%, which was 6.5% higher than that obtained with free agarases. After eight cycles, the multienzyme biocatalyst still preserved 46.4% of the initial activity. To the best of our knowledge, this is the first report where two different agarases were coimmobilized. These results demonstrated the feasibility of the new method to fabricate a new multienzyme system onto magnetic nanoparticles via covalent bonds to produce l-AHG

Low-Temperature Reversible Hydrogen Storage Properties of LiBH₄: A Synergetic Effect of Nanoconfinement and Nanocatalysis

LiBH₄ has been loaded into a highly ordered mesoporous carbon scaffold containing dispersed NbF₅ nanoparticles to investigate the possible synergetic effect of nanoconfinement and nanocatalysis on the reversible hydrogen storage performance of LiBH₄. A careful study shows that the onset desorption temperature for nanoconfined LiBH₄@MC-NbF₅ system is reduced to 150 °C, 225 °C lower than that of the bulk LiBH₄. The activation energy of hydrogen desorption is reduced from 189.4 kJ mol^–1 for bulk LiBH₄ to 97.8 kJ mol^–1 for LiBH₄@MC-NbF₅ sample. Furthermore, rehydrogenation of LiBH₄ is achieved under mild conditions (200 °C and 60 bar of H₂). These results are attributed to the active Nb-containing species (NbH_<i>x</i> and NbB₂) and the function of F anions as well as the nanosized particles of LiBH₄ and high specific surface area of the MC scaffold. The combination of nanoconfinement and nanocatalysis may develop to become an important strategy within the nanotechnology for improving reversible hydrogen storage properties of various complex hydrides

<i>In vivo</i> MRI and pathology of the three groups.

<p>T2*-weighted imaging was used in the three groups (n = 5, respectively). The signal intensities of the kidneys decreased significantly in the targeting group after injection (b, white arrow) compared to before injection (a). Diffusive iron particle depositions in the glomerulus (c, 400×, black arrow) and a few iron particles deposited in renal tubules (c, 400×, right corner) are demonstrated. TEM confirmed iron particles (d, 30000×, blue arrow) deposited under foot process. No significant signal intensity changes were observed in the kidneys of the untargeted and control groups between before and after injection. Iron particle depositions in the glomeruli of the untargeted group (g, 400×) and control group (k, 400×) were not observed. However, several iron particles were deposited in some renal tubules (g and k, yellow triangle) in both groups. TEM confirmed the absence of iron particle deposition under foot process (h and l, 12500×).</p

Samples of the ROIs.

<p>T2*WI pictures of a kidney in targeting group are shown. a, before injection. b, after injection. Significant signal decrease of the cortex was observed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121244#pone.0121244.g003" target="_blank">Fig. 3B</a>. The cortex, outer medulla, and inner medulla were measured in proper order from the outer compartment to inner compartment.</p

The changes in rSI (ΔrSI) of the three groups.

<p>The rSI decreased remarkably in targeting group compared to the other two groups. The differences in ΔrSI between the targeting group and untargeted group or control group were also statistically significant (*: <i>p</i><0.05, v.s. targeted group).</p

Pathology for HN model and normal rats.

<p>Histological images (HE staining, 400×) showed no obvious changes in glomerulus in HN rats (a) compared to normal rats (d). TEM (12500×) images of the kidneys showed segmental thickening of the GBM (b, black asterisk) and foot process fusion (b, white asterisk) in an HN rat. GBM (e, black asterisk) thickening and foot process (e, white asterisk) fusion were not observed in normal rats. Immunofluorescence showed C5b-9 depositions along the GBM in a glomerulus of HN rats (c), and no C5b-9 deposition in normal rats (f).</p

Histograms of the signal intensity distributions in one case of the targeting group.

<p>The average MR unit values of the cortex (ROI 1), outer medulla (ROI 2) and inner medulla (ROI 3) decreased significantly after probe injection. X-axis, MR unit values of pixels. Y-axis, counts of pixels.</p

Three animal groups and brief experimental steps.

<p>Tweleve HN rats were randomly divided into targeting and untargeted groups (n = 6 in each group). Five normal SD rats were used as the control group. Anti-C5b-9-USPIO was intravenously injected into the targeting and control groups, and nonspecific IgG-USPIO was injected into the untargeted group. MRI sessions were performed before injection and 24 hours after injection.</p

Biochemical parameters in urine and plasma of the model group and nomal group.

<p>Biochemical parameters in urine and plasma of the model group and nomal group.</p

Synergetic Effect of in Situ Formed Nano NbH and LiH_1–<i>x</i>F_<i>x</i> for Improving Reversible Hydrogen Storage Properties of the Li–Mg–B–H System

A significant improvement in hydrogenation/dehydrogenation properties of 2LiH/MgB₂ can be achieved by adding NbF₅. The results show that the NbF₅ additive is effective for enhancing the de/hydrogenation kinetics of the Li–Mg–B–H system and reducing the desorption temperatures of MgH₂ and LiBH₄. For the 2LiH–MgB₂–0.03NbF₅ sample, About 9.0 wt % hydrogen capacity is obtained rapidly under cyclic conditions of rehydrogenation within 20 min at 350 °C and dehydrogenation within 20 min at 400 °C; thus, catalytic improvement persists well in the subsequent reversible dehydrogenation cycles. Moreover, the sample could reversibly reabsorb and release more than 9.0 wt % hydrogen even at 250 and 375 °C, respectively. Microstructure analyses reveal that the NbF₅ additive in improving the de/hydrogenation properties of Li–Mg–B–H system could be ascribed to the synergistic effect of in situ formed nano NbH particles acting as “active gateways” facilitating the diffusion of hydrogen, and the “favorable thermodynamic destabilization” from the reversible transition of LiH_1–<i>x</i>F_<i>x</i> caused by functionality of F-anion substitution. This fundamental understanding provides us with insights into the design and optimization of the catalytic method and species for the catalyzed Li–Mg–B–H system