The research team, led by Yijie Liu and Jie Teng, worked with fresh leaves of the ‘Gougunao Tea No. 2’ cultivar harvested in Jiangxi Province, China. Their target was a purification strategy called three-phase partitioning, or TPP, a chromatography-free separation technique in which t-butanol and water, normally miscible, are forced apart by adding ammonium sulfate. Under the right conditions, proteins gather into a distinct middle phase between the upper organic layer, which collects oils and lipid-soluble material, and the lower aqueous layer, which retains polysaccharides and other water-soluble compounds. TPP is prized for its simplicity, high recovery rates and easy scalability, and it has already been applied to polyphenol oxidase from several sources. The problem is the solvent: t-butanol is flammable, volatile and ecotoxic, and highly polar organic solvents can disrupt the hydrophobic interactions that hold membrane proteins in their native shape, accelerating the loss of enzyme activity during purification.

To replace t-butanol, the researchers turned to deep eutectic solvents, a class of liquids first described by Abbott and colleagues in 2004. Deep eutectic solvents form when a hydrogen bond acceptor and a hydrogen bond donor self-assemble through an extensive hydrogen bonding network, producing a liquid with a melting point far below that of either component. They are simple to prepare, biodegradable, low in toxicity and highly biocompatible, and their physicochemical properties can be tuned by choosing different component pairs. Previous work has shown that these solvents can stabilize proteins in vitro through hydrogen bonding and hydrophobic forces, which made them attractive candidates for a green version of TPP. However, no one had applied a deep eutectic solvent-based TPP system to a membrane-bound polyphenol oxidase, and the molecular mechanisms behind any such purification remained unexplored.

The team prepared nine different deep eutectic solvents from combinations of hydrogen bond acceptors and donors, including pairs such as tetracaine with thymol, menthol with octanoic acid, and procainamide derivatives with fatty acids. Fourier transform infrared spectroscopy confirmed that each mixture had formed a genuine eutectic: carbonyl stretching peaks shifted to lower frequencies, and the broad hydroxyl absorption bands of the individual components merged into single strong peaks, signatures of newly created intermolecular hydrogen bonds. Proton nuclear magnetic resonance spectroscopy provided further confirmation. The phenolic hydroxyl proton of thymol in the first solvent shifted downfield with clear peak broadening, while the carboxyl protons of several acid-based mixtures vanished entirely from the 8 to 15 ppm region, a consequence of rapid proton exchange within a dense hydrogen bonding network. One solvent, built from menthol and octanoic acid in a one-to-one molar ratio and designated DES-5, showed the largest downfield shift of all, indicating the strongest hydrogen bonding in the entire set.

When the nine solvents were tested against crude tea mPPO extract, DES-5 stood out decisively. It delivered an enzyme activity of 2117 units and a purification fold of 17.43, with a specific activity of 50,652 units per milligram, significantly outperforming t-butanol and every other solvent tested. The authors attribute this to a combination of favorable mass transfer properties and a useful side effect: the solvent appears to denature some contaminating proteins, which raises the purification fold of the target enzyme. Single-factor experiments then mapped how the process responded to changing conditions. Enzyme activity and purification fold both peaked at 45 percent ammonium sulfate, a concentration high enough to salt out proteins through hydrophobic aggregation but low enough to avoid irreversible conformational damage. The optimal solvent-to-extract ratio was 2:1 by volume, beyond which excess solvent began to denature the enzyme itself. Extraction time peaked at 75 minutes, after which prolonged exposure to air and light caused oxidative losses, and the ideal temperature was 30 degrees Celsius, where the lower viscosity of the warm solvent improved mass transfer before thermal inactivation set in.

Response surface methodology refined these parameters into a precise recipe. A Box-Behnken design produced quadratic regression models for both enzyme activity and purification fold, with coefficients of determination of 0.9966 and 0.9992 respectively, indicating excellent fit and predictive reliability. The statistically optimal conditions were 47.2 percent ammonium sulfate, 74.8 minutes of extraction and a solvent ratio of 1.98:1. Rounded to practical values of 47 percent, 75 minutes and 2:1, three replicate validations yielded an average activity of 1967 units and a purification fold of 16.64, with prediction errors below one percent and no statistically significant difference from the theoretical values. The optimized process was thus both accurate and reproducible.

Physical characterization reinforced the case for the green solvent. Scanning electron microscopy revealed that crude mPPO formed irregular porous aggregates with rough edges and abundant protein debris. After t-butanol-based purification, the particles became dense, fragmented shards, a morphology the authors link to organic-solvent-induced aggregation. mPPO purified through DES-5, by contrast, emerged as relatively intact block-shaped particles with denser, smoother surfaces and minimal fragmentation, suggesting the deep eutectic solvent provides a gentler separation environment that preserves the morphological integrity of the protein particles.

To understand the mechanism, the researchers built a homology model of tea mPPO from its UniProt sequence, validated it with Ramachandran and ERRAT analyses, and docked the two DES-5 components into the structure. Both menthol and octanoic acid bound with energies more favorable than the minus 5 kilocalories per mole threshold for spontaneous binding. Menthol’s single hydroxyl group acted as both hydrogen bond donor and acceptor, bridging three backbone residues, Gly301, Lys382 and Gly383, with bond lengths between 2.2 and 2.6 angstroms. Octanoic acid anchored its polar head group through hydrogen bonds to Leu531, Thr331 and Asn200, the shortest at just 1.9 angstroms, while surrounding hydrophobic residues constrained its fatty acyl chain and improved shape complementarity within the binding pocket.

Molecular dynamics simulations in GROMACS then tested whether these interactions stabilize or destabilize the enzyme. In three independent 100-nanosecond simulations per system, the protein backbone of both ligand-bound complexes relaxed and stabilized at root-mean-square deviations of roughly 0.20 to 0.26 nanometers, whereas an additive-free control lacking both ligands never reached a plateau and drifted to 0.30 to 0.35 nanometers, clear evidence that menthol and octanoic acid dampen backbone fluctuations. The dicopper catalytic center, restrained to preserve its native type 3 copper geometry, kept its bridging angle centered at 120 to 130 degrees throughout, confirming that ligand binding does not disturb the active site. The radius of gyration held steady at about 2.22 nanometers in both systems, showing no global unfolding, and free energy landscape analysis revealed funnel-shaped topologies converging on compact, low-energy states. In the menthol system the solvent-accessible surface area rose modestly as surface loops rearranged outward, while in the octanoic acid system it decreased as the ligand shielded surface residues, producing a more compact surface conformation.

Taken together, the experiments and simulations tell a coherent story: a menthol and octanoic acid deep eutectic solvent purifies tea membrane-bound polyphenol oxidase more efficiently than t-butanol precisely because its components bind the enzyme gently, through hydrogen bonds and hydrophobic contacts that stabilize rather than disrupt the protein’s native fold. The optimized protocol, 47 percent ammonium sulfate, 75 minutes, a 2:1 solvent ratio and 30 degrees Celsius, achieves a purification fold above 16 in a single chromatography-free step using a biodegradable solvent. Beyond tea, the authors suggest the approach offers a template for the green purification of other membrane-bound enzymes, a class of catalysts central to cellular processes and industrial biocatalysis but long hindered by purification methods that sacrifice the very activity they seek to recover.

Subject of Research: Green purification of tea membrane-bound polyphenol oxidase using deep eutectic solvent-based three-phase partitioning

Article Title: Molecular dynamics provide insights into the purification of tea membrane-bound polyphenol oxidase through three-phase partitioning utilizing an eco-friendly deep eutectic solvent

Article References: Liu, Y., Hou, S., Xiao, S., Wang, Y., Liu, Y., & Teng, J. (2026). Molecular dynamics provide insights into the purification of tea membrane-bound polyphenol oxidase through three-phase partitioning utilizing an eco-friendly deep eutectic solvent. Food Chemistry: X, 39, Article 104529. https://doi.org/10.1016/j.fochx.2026.104529

Image Credits: AI Generated

DOI: 10.1016/j.fochx.2026.104529

Keywords: polyphenol oxidase, deep eutectic solvent, three-phase partitioning, tea, molecular dynamics, enzyme purification, green chemistry, membrane proteins, molecular docking, food chemistry, biocatalysis, protein stability