ABSTRACT
Background: A simple and efficient strategy for agarase immobilization was developed with carboxyl-functionalized magnetic nanoparticles (CMNPs) as support. The CMNPs and immobilized agarase (agarase-CMNPs) were characterized by transmission electron microscopy, dynamic light scattering, vibrating sample magnetometry, scanning electron microscopy, X-ray diffraction, thermogravimetric analysis, and zeta-potential analysis. The hydrolyzed products were separated and detected by ESI-TOF-MS. Results: The agarase-CMNPs exhibited a regular spherical shape with a mean diameter of 12 nm, whereas their average size in the aqueous solution was 43.7 nm as measured by dynamic light scattering. These results indicated that agarase-CMNPs had water swelling properties. Saturation magnetizations were 44 and 29 emu/g for the carriers and agarase-CMNPs, respectively. Thus, the particles had superparamagnetic characteristics, and agarase was successfully immobilized onto the supports. Agaro-oligosaccharides were prepared with agar as substrate using agarase-CMNPs as biocatalyst. The catalytic activity of agarase-CMNPs was unchanged after six reuses. The ESI-TOF mass spectrogram showed that the major products hydrolyzed by agarase-CMNPs after six recycle uses were neoagarotetraose, neoagarohexaose, and neoagarooctaose. Meanwhile, the end-products after 90 min of enzymatic treatment by agarase-CMNPs were neoagarobiose and neoagarotetraose. Conclusions: The enhanced agarase properties upon immobilization suggested that CMNPs can be effective carriers for agarase immobilization. Agarase-CMNPs can be remarkably used in developing systems for repeated batch production of agar-derived oligosaccharides.
Subject(s)
Oligosaccharides/metabolism , Enzymes, Immobilized , Magnetite Nanoparticles/chemistry , Glycoside Hydrolases/metabolism , Thermogravimetry , X-Ray Diffraction , Enzyme Stability , Catalysis , Microscopy, Electron, Transmission , Magnetometry , Dynamic Light Scattering , Glycoside Hydrolases/chemistryABSTRACT
Objective: To study the inhibition effect of agaro-oligosaccharide (AOS) on neovascularization and its mechamism. Method: The anti-angiogenic effect in vivo was evaluated on chicken chorioallantoic membrane (CAM) model. Cytotoxic activity of AOS was demonstrated by inhibition of several human cell lines by MTT assay. The apoptosis of HUVECs induced by AOS was examined by Hoechst staining assay and quantified by flowcytometry. Results: In CAM, AOS caused dose-dependent decrease on the vascular density and adversely affected capillary plexus formation. Different cytotoxic sensitivities were observed for AOS towards several kinds of cell lines, and HUVECs were more sensitive. Moreover, the growth inhibitory activity was correlated with induction of apoptosis. Flowcytometric analysis also revealed that AOS arrested the cell cycle progression at S phase. Conclusion: Agaro-oligosaccharide possesses the anti-angiogenic effects, which are associated with apoptosis induction of endothelial cells.