Nitrilotriacetic Acid Functionalized Microgels for Efficient Immobilization of Hyaluronan Synthase

Enzymes play a vital role in synthesizing complex biological molecules like hyaluronic acid (HA). Immobilizing enzymes on support materials is essential for their efficient use and reuse in multiple cycles. Microgels, composed of cross-linked, highly swollen polymer networks, are ideal for enzyme uptake owing to their high porosity. This study demonstrates the immobilization of His6-tagged hyaluronan synthase from Pasteurella multocida (PmHAS) onto nitrilotriacetic acid functionalized microgels using different bivalent ions (Ni2+, Co2+, Mn2+, Mg2+, and Fe2+) via metal affinity binding. The results indicate that using Ni2+ yields the microgels with the highest enzyme uptake and HA formation. The immobilized PmHAS enables repetitive enzymatic production, producing high molecular weight HAs with decreasing dispersities in each step. Furthermore, the highest reported yield of HA with high molecular weight for immobilized PmHAS is achieved. This system establishes a foundation for continuous HA formation, with future works potentially enhancing PmHAS stability through protein engineering.Enzymes are crucial for synthesizing complex molecules such as hyaluronic acid (HA). In this work, the advantages of enzyme immobilization are showcased by binding His6-tagged hyaluronan synthase from Pasteurella multocida to microgels through affinity binding. This process facilitates repetitive HA production, resulting in the highest yield to date using immobilized PmHAS. Thus, a foundation for continuous HA formation is established. image