Exolytic products of alginate by the immobilized alginate lyase confer antioxidant and antiapoptotic bioactivities in human umbilical vein endothelial cells.

Alginate is a natural polysaccharide resource abundant in brown algae and it can be cleaved into alginate oligosaccharides by alginate lyase. Alginate lyases and the bioactive alginate oligosaccharides have been applied in diverse fields such as pharmaceutical therapy and nutraceutical supplementation. Immobilized enzymes greatly facilitate their industrial application owing to their reusability, stability, and tunability. In this study, magnetic Fe3O4 nanoparticles were synthesized and used to immobilize an exolytic alginate lyase AlgL17 that was characterized previously. The immobilized AlgL17 demonstrated enhanced thermal and pH tolerance, extended storage stability, and moderate reusability. The mass spectrum indicated the specific activity of the immobilized AlgL17 to release alginate oligosaccharides (AOS) from alginate polysaccharide. The produced AOS exhibited their antioxidant and antiapoptotic activities in H2O2-stressed human umbilical vein endothelial cells by upregulation of reactive oxygen species scavenging activities and attenuation of the caspase-mediated apoptosis pathway.

Molecular mechanism of Aspergillus fumigatus biofilm disruption by fungal and bacterial glycoside hydrolases.

During infection, the fungal pathogen Aspergillus fumigatus forms biofilms that enhance its resistance to antimicrobials and host defenses. An integral component of the biofilm matrix is galactosaminogalactan (GAG), a cationic polymer of α-1,4-linked galactose and partially deacetylated N-acetylgalactosamine (GalNAc). Recent studies have shown that recombinant hydrolase domains from Sph3, an A. fumigatus glycoside hydrolase involved in GAG synthesis, and PelA, a multifunctional protein from Pseudomonas aeruginosa involved in Pel polysaccharide biosynthesis, can degrade GAG, disrupt A. fumigatus biofilms, and attenuate fungal virulence in a mouse model of invasive aspergillosis. The molecular mechanisms by which these enzymes disrupt biofilms have not been defined. We hypothesized that the hydrolase domains of Sph3 and PelA (Sph3h and PelAh, respectively) share structural and functional similarities given their ability to degrade GAG and disrupt A. fumigatus biofilms. MALDI-TOF enzymatic fingerprinting and NMR experiments revealed that both proteins are retaining endo-α-1,4-N-acetylgalactosaminidases with a minimal substrate size of seven residues. The crystal structure of PelAh was solved to 1.54 Å and structure alignment to Sph3h revealed that the enzymes share similar catalytic site residues. However, differences in the substrate-binding clefts result in distinct enzyme-substrate interactions. PelAh hydrolyzed partially deacetylated substrates better than Sph3h, a finding that agrees well with PelAh's highly electronegative binding cleft versus the neutral surface present in Sph3h Our insight into PelAh's structure and function necessitate the creation of a new glycoside hydrolase family, GH166, whose structural and mechanistic features, along with those of GH135 (Sph3), are reported here.