Ice-binding protein's dual roles for prolonging the enzymatic activity of alcohol dehydrogenase against freezing
Year: 2025
Type: article
Abstract: 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. • Ice-binding protein (DAFP-1) effectively protects alcohol dehydrogenase (ADH) activity. • Ice recrystallization inhibition (IRI) of DAFP-1 is more potent than its cryoprotective effects. • Enzymatic activity and fluorescence assays implicate DAFP-1 near ADH's active site. • Docking/MD support the dual role of DAFP-1 in cryoprotection: IRI plus molecular interactions.
Authors: Daisy Aguirre, Isabella Nieblas, Alfredo Gonzalez, Emily L. Zempel, Brenda A. Rounds, Ting-Yu Wang, Tsui-Fen Chou, Ashley Cheng, Jaelyn Asamoah, Kenneth Liu, Aidan Reyes, Caitlin E. Scott, Xin Wen
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $4,390
Awards: G4654614292, REU Site: Research Experience for Undergraduates in Chemistry and Biochemistry, Equipment: MRI: Track 1 Acquisition of Expansion of the Molecular Education and Research Consortium in Undergraduate Computational Chemistry (MERCURY), PREC Track 1: Cal State LA - MolSSI PREC Pathway to Diversity Program, IDENTIFICATION AND MOLECULAR BASIS FOR EFFICIENT ANTIFREEZE PROTEIN ENHANCERS, MRI: Expansion of the Molecular Education and Research Consortium in Undergraduate Computational ChemistRY (MERCURY) via Addition of High Performance Computers, G9000060866, G9000108296, Bridges to the Doctorate Program at Cal State LA
OpenAlex ID: W4416768266