Functional characterization and effect of polysaccharides on the activity of GH16 β-agarase from Gelidibacter...

β-agarase is the key enzyme for the primary degradation of agar, a major component of red algae. This study demonstrates cloning, overexpression and characterization of a recombinant GH16 β-agarase (WP_199597959) from Antarctic bacterium Gelidibacter salicanalis PAMC21136. Biochemical properties demonstrated that the maximum activity was observed at pH and temperature of 7.0 and 50°C, respectively using agarose as a substrate. The agarolytic activity of WP_199597959 on agarose was found to be endo-type with production of neoagarobiose (NA2) as a major product. Mn²⁺ metal ion significantly enhanced the activity, doubling the reaction rate as compared with other metal ions. Neoagarooligosaccharides (NAOSs) like neoagarotetraose (NA4), and neoagarohexaose (NA6) hydrolyzed into NA2. In-silico analysis demonstrated that the key catalytic residues E184 and E189 are involved in retaining hydrolytic mechanisms. In-vitro, interaction with negatively charged carboxymethyl cellulose (CMC) enhanced the enzyme activity by 15% while positively charged chitosan (CS) decreased the enzyme activity by 12%, highlighting the effect of biopolymers on enzyme activity within the microenvironment interaction. These findings may provide insight into biochemical properties and application of WP_199597959 for agarose degradation.