Odd-Numbered Agaro-Oligosaccharides Produced by α-Neoagaro-Oligosaccharide Hydrolase Exert Antioxidant Activity in Human Dermal Fibroblasts.

Abstract

Agarases produce agar oligosaccharides with various structures exhibiting diverse physiological activities. α-Neoagaro-oligosaccharide hydrolase (α-NAOSH) specifically cleaves even-numbered neoagaro-oligosaccharides, producing 3,6-anhydro-l-galactose (l-AHG) and odd-numbered agaro-oligosaccharides (OAOSs). In this study, α-NAOSH from the agar-degrading marine bacterium Gilvimarinus agarilyticus JEA5 (Gaa117) was purified and characterized using an E. coli expression system to produce OAOSs and determine their bioactivity. Recombinant Gaa117 (rGaa117) showed maximum activity at pH 6.0 and 35 °C. rGaa117 retained >80% of its initial activity after 120 min at 30 °C. The activity was enhanced in the presence of Mn²⁺. K_m, V_max, and K_cat/K_m values of the enzyme were 22.64 mM, 246.3 U/mg, and 15 s^-1/mM, respectively. rGaa117 hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose, producing OAOSs that commonly contained l-AHG. Neoagarobiose and neoagarotetraose mixtures, designated NAO24, and mixtures of l-AHG and agarotriose, designated AO13, were obtained using recombinant rGaa16B (β-agarase) and rGaa117, respectively, and their antioxidant activities were compared. AO13 showed higher hydrogen peroxide-scavenging activity than NAO24 in human dermal fibroblasts in vitro because of structural differences: AOSs have d-galactose at the non-reducing end, whereas NAOSs have l-AHG. In conclusion, OAOSs exhibited high ROS-scavenging activity in H₂O₂-induced human dermal fibroblasts. They may be applicable in cosmetics and pharmaceuticals for prevention of skin aging.