The hyaluronidase inhibitory effect of partial hydrolysates of Agar by a novel β-Agarase from Gilvimarinus agarilyticus JEA5

Abstract

Agar is the main component of the cell wall in several species of red seaweed. Agar is widely used as a gelling substance in various industries. In addition to these classical applications, many agar-derived oligosaccharides exhibit various biological and therapeutic properties. In this study, we discovered that gaa16b, a gene isolated from the marine bacterium Gilvimarinus agarilyticus JEA5, encodes a novel β-agarose. We cloned the gaa16b gene and overexpressed it as a recombinant protein in E. coli BL21(DE3). Next, maltose-binding protein (MBP)-tagged Gaa16B was purified and analyzed to investigate its biochemical properties and hydrolytic patterns. Additionally, to validate the potential of the partial hydrolytic product of recombinant Gaa16B (rGaa16B) for cosmetic use, we carried out a hyaluronidase inhibitory assay. rGaa16B showed the optimal temperature and pH at 55℃ and pH 6-7, respectively, and the protein was highly stable at 55℃ for 90 min. additionally, rGaa16B activity was strongly enhanced (2.3-fold) in the presence of 2.5 mM MnCl2. The Km and Vmax of rGaa16B for agarose were 6.4 mg/mL and 963 U/mg, respectively. Thin layer chromatography analysis revealed that rGaa16B can hydrolyze agar into neoagarotetraose and neoagarobiose. Partial hydrolysis product (PHPs) of rGaa16B had an average molecular weight of 88-102 kDa and exhibited <60% hyaluronidase inhibition activity at a concentration of 1 mg/mL, whereas completely hydrolysis product (CHP) showed no hyaluronidase at the same concentration The bio-chemical properties of Gaa16B suggest that it could be useful for producing functional neoagarooligosaccharides. Additionally, the PHP of rGaa16B may be useful in promoting its utilization which is limited due to the gel strength of agar.