Abstract
We recently identified a β-agarase, Gaa16B, in the marine bacterium Gilvimarinus agarilyticus JEA5. Gaa16B, belonging to the glycoside hydrolase 16 family of β-agarases, shows less than 70.9% amino acid similarity with previously characterized agarases. Recombinant Gaa16B lacking the carbohydrate-binding region (rGaa16Bc) was overexpressed in Escherichia coli and purified. Activity assays revealed the optimal temperature and pH of rGaa16Bc to be 55 °C and pH 6–7, respectively, and the protein was highly stable at 55 °C for 90 min. Additionally, rGaa16Bc activity was strongly enhanced (2.3-fold) in the presence of 2.5 mM MnCl2. The Km and Vmax of rGaa16Bc for agarose were 6.4 mg/mL and 953 U/mg, respectively. Thin-layer chromatography analysis revealed that rGaa16Bc can hydrolyze agarose into neoagarotetraose and neoagarobiose. Partial hydrolysis products (PHPs) of rGaa16Bc had an average molecular weight of 88–102 kDa and exhibited > 60% hyaluronidase inhibition activity at a concentration of 1 mg/mL, whereas the completely hydrolyzed product (CHP) showed no hyaluronidase at the same concentration. The biochemical properties of Gaa16B suggest that it could be useful for producing functional neoagaro-oligosaccharides. Additionally, the PHP of rGaa16Bc may be useful in promoting its utilization, which is limited due to the gel strength of agar.
Highlights
Introduction published maps and institutional affilAgar is the main component of the cell wall in several species of red seaweed and consists of a mixture of two polysaccharides: agarose and agaropectin
The deduced amino acid sequence of Gaa16B showed the highest identity (90.7%) and similarity (93.8%) with Gilvimarinus polysaccharolyticus (WP_049721028.1), whereas it exhibited less than 55.9% identity and 70.9% similarity with functionally characterized agarases (Table 1)
The enzymatic reactions for calculating Km and Vmax values were performed under optimal conditions, and the results showed that the Km, Vmax, and Kcat values of rGaa16Bc for agarose were 6.4 mg/mL, 953 U/mg, and 201.2 s−1, respectively. 4 of 12
Summary
Introduction published maps and institutional affilAgar is the main component of the cell wall in several species of red seaweed and consists of a mixture of two polysaccharides: agarose and agaropectin. Agar is widely used as a gelling substance in the pharmaceutical, cosmetic, and food industries, as well as in the life sciences, where it is used in a range of techniques, including microbiome culturing media, electrophoresis gel, and chromatography resin. In addition to these classical applications, many agar-derived oligosaccharides exhibit various biological and therapeutic properties and, have the potential for diverse applications in the cosmetic, medicinal, and pharmaceutical industries [2,3]. Based on the mechanism of their actions, agarases are categorized into two main groups, namely, α-agarases and β-agarases. αagarases hydrolyze α-1,3-linkages between the composite sugars in agarose to generate iations
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