Interactions of Veterinary Antibiotics with Purified Bovine Milk Alkaline Phosphatase: A Combined In Vitro Kinetic and Theoretical DFT/HF/M06-2X Study.

Abstract

Alkaline phosphatase (AP, EC 3.1.3.1) is a key metalloenzyme and has been widely used as a reliable marker for milk pasteurization. Milk containing antibiotic residues tends to inhibit AP function, which can result in faulty pasteurization determination. AP from bovine milk was purified by a purification strategy involving butanol homogenization, rennet precipitation, and Sephadex G-100 gel filtration chromatography. The enzyme was purified 53.43-fold with a specific activity of 7516.40 EU/mg. Kinetic characterization showed a K_M value of 2.27 mM (V_max value: 555.56 EU/min); substrate was p-nitrophenyl phosphate. Electrophoretic studies verified the purity and molecular nature of the product enzyme. To functionally validate the identity of the purified enzyme, tetramisole hydrochloride was evaluated as a standard reference inhibitor, yielding a potent IC₅₀ value of 0.045 mM. Fifteen veterinary drugs and antibiotics were used to test the inhibitory effects of purified milk AP in vitro. Among the antibiotic compounds tested, Danofloxacin Mesylate induced the strongest antibiotic inhibition (IC₅₀: 3.36 mM). Gentamycin Sulfate, Metamizole Sodium, Enrofloxacin, Kanamycin Sulfate, Sulfadimidine Sodium, and Neomycin Sulfate had IC₅₀ values ranging between 22.89 and 312.13 mM of inhibition in AP, while Dexamethasone, Oxytetracycline Hydrochloride, Marbofloxacin, Tilmicosin, Doxycycline Monohydrate, Tylosin, Florfenicol and Flunixin Meglumine made no detected inhibition in the presence of 0.5 mM p-nitrophenyl phosphate. The molecular docking studies were additionally performed that the density functional theory (DFT), Hartree-Fock (HF), and M06-2X calculations were used to validate the experimental results with the 6-31++G(d,p) basis set. The theoretical results also reported that Oxytetracycline Hydrochloride, Tilmicosin, and Doxycycline Monohydrate displayed superior electronic reactivity, lower energy gaps between HOMO-LUMO, and more electrophilic character compared with the other compounds. These results are the first complete assessment of the impact of veterinary antibiotics in milk AP and underscore the possible role of antibiotic residues in establishing the reliability of pasteurization control measures.