(174ca) Site-Specific Covalent Immobilization of ?-Agarase Onto Magnetic Nanoparticles for the Conversion of Gelidium Amansii into Biologically-Active Sugars

Red macroalgae Gelidium amansii is considered as a prospective renewable resource due to its ubiquity, high carbohydrate and low lignin contents. Its cell wall is rich with carbohydrates such as agarose, and agarose-derived neoagarooligosaccharides (NAOSs): neoagarohexaose (NA6), neoagarotetraose (NA4) and neoagarobiose (NA2), which have various industrial potentials due to their various physiological activities i.e., anticariogenic, antiinflammatory, skin whitening and moisturizing properties that can be applied in the medical, pharmaceutical and cosmetics industries. Selective production of these sugars without degradation is needed to preserve their bio-functionality. In this context, enzymatic hydrolysis is highly desirable as this process: avoids sugar degradation and use of expensive reactors, is less energy-intensive, involves benign reaction conditions (i.e. physiological pH, temperature and pressure), and produces less toxic hydrolysates to the downstream processes. However, this technique remains infeasible due to the high enzyme cost and lack of thermal stability of free β-agarase enzymes, which perform the hydrolysis at a temperature above the sol-gel transition temperature of agarose. In this study, the controlled, site-specific and covalent cross-linking of an engineered β-agarase (Aga2 from Cellulophaga omnivescoria W5C) was successfully carried out on amine-functionalized magnetic nanoparticles (NPs). Five tyrosine residues (Y-tag) were introduced at the N-terminus of the enzyme through genetic engineering, and mushroom tyrosinase was utilized as catalyst for the crosslinking and immobilization of the enzyme. Overall results indicate that site-specific immobilization of β-agarase improved its thermal stability and while the use of magnetic NP support ensured its recyclability for repeated production of NAOSs from Gelidium amansii.

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2018R1D1A1B07043993, No. 2018R1D1A1B07048107 and 22A20130012051(BK21Plus)) and by the Ministry of Science and ICT(2016R1C1B1013252).