Ice-binding protein's dual roles for prolonging the enzymatic activity of alcohol dehydrogenase against freezing.

As biological catalysts, enzymes are essential not only in life, but also various industrial processes. Preservation of enzymatic activity against freeze-thaw damage is critical for many industrial applications. As a type of ice-binding proteins, antifreeze proteins (AFPs) are notable for their ability to control ice growth and ice recrystallization inhibition (IRI). Additionally, AFPs were recently discovered to interact with sugars, nucleosides, and other proteins, making them remarkably attractive for biopreservation. Here, we characterize the role of DAFP-1, a recombinant insect AFP in preserving the activity of industrially important alcohol dehydrogenase (ADH) in dilute solutions at temperatures below freezing. The presence of DAFP-1 at a molar ratio of 4:1 effectively preserves ADH enzyme activity following multiple freeze-thaw cycles. The concentration of DAFP-1 for its IRI activity is substantially lower than that for optimal ADH activity preservation. Enzymatic assays and fluorescence spectroscopy suggest DAFP-1's effects on ADH's conformation around the Trp-His pairs close to its catalytic regions. The interaction between DAFP-1 and ADH is further elucidated by molecular docking and corroborated by molecular dynamics simulations. These findings reveal an AFP's dual protective roles in effective enzyme cryoprotection, highlighting the importance of molecular interactions in cryopreservation by AFPs for bioinspired design.