Tailoring Casein Functionality via Protein Glutaminase-Mediated Deamidation: Insights from Recombinant Enzyme Production and Structural Analysis

Casein, a major milk protein, faces limited industrial applications due to its hydrophobicity. Traditional chemical modifications (e.g., sodium caseinate) often compromise nutritional value, while enzymatic deamidation using protein glutaminase (PG) is hindered by low enzyme yields and endotoxin risks from conventional Escherichia coli expression systems. Here, we developed a safe and efficient strategy by expressing PG in probiotic E. coli Nissle 1917 (EcN), eliminating endotoxin production. The recombinant PG yielded 8.69 U/mL (1.64-fold higher than control) and retained stability at ≤ 50 ℃ and pH 4-8. Casein was deamidated under mild conditions (50 ℃, pH 7, 180 min) with a low enzyme-to-substrate ratio (5 U/g protein), reaching a 54.4% degree of deamidation (DD). Deamidation induced structural reorganization: β-sheet content decreased by 44.1%, while α-helix and β-turn increased by 22.5% and 24.4%, respectively. This exposed hydrophobic residues (e.g., tyrosine), reduced particle size to 200 nm, and enhanced electrostatic repulsion (zeta potential: −27.4 mV vs. −21.4 mV for undeamidated casein). Consequently, solubility surged from 1.2% to 80.5% under neutral conditions, with 1.8-fold improved foaming, 1.4-fold and 7.3-fold higher emulsifying property and emulsifying stability compared to untreated casein, respectively. Deamidation improved in vitro digestibility (77.4% gastrointestinal digestibility) and amino acid balance, alleviating leucine limitation and increasing the essential amino acid index to 100.1. This study pioneers the use of EcN for endotoxin-free PG production and demonstrates that enzymatic deamidation outperforms chemical methods by avoiding hydrolysis and toxicity and preserving functionality. The modified casein is directly applicable to dairy products (e.g., yogurt, cheese) as a natural emulsifier or foaming agent, offering a sustainable alternative to synthetic additives.