Enzymatic dephosphorylation of isolated bovine caseins: Targeted modulation of phosphorylation degree via pH and reaction time optimization

This study investigated the effect of dephosphorylation conditions for resulting phosphorylation degrees of isolated bovine α S - (α S1 - + α S2 -), β-, and κ-caseins (CNs), defining routes for obtaining caseins with targeted phosphorylation degrees. After the enzymatic dephosphorylation reactions by bovine alkaline phosphatase (BAP) at different conditions, the resulting phosphorylation degrees were comprehensively characterized using LC-ESI/MS Single Quadrupole analysis. Adjustments in pH and reaction time facilitated distinct phosphorylation states while balancing the extent of casein degradation, achieving complete dephosphorylation within 3 to 6 h when choosing optimal conditions. Specifically, enzymatic dephosphorylation of α S -CN at pH 8.5 for 30 s resulted in proteoforms with 4–6 phosphate groups being predominant, whereas over 90% of α S -CN was non-phosphorylated after 6 hours, with limited degradation observed. Similarly, for the dephosphorylation processes of β-CN or κ-CN at pH values 7.0 or 8.5, respectively, beyond 3 h led to complete dephosphorylation with over 90% of these caseins containing no phosphate groups, accompanied by minimal degradation. Controlled modulation of phosphorylation degrees constitutes a crucial experimental foundation for establishing structure-function relationships between phosphate content and casein functionalities, guiding the engineering of casein ingredients with targeted applications for both bovine- and microbial-derived systems. • Enabled tunable phosphorylation degrees of α S -, β-, and κ-CNs via BAP treatment • Determined isoform-specific optimal pH: 8.5, 7.0, and 8.5 for α S -, β-, and κ-CN • Defined pH and time thresholds balancing phosphate removal and casein integrity • Achieved 90–100% phosphate removal of α S /β/κ-CN in 6/3/3 h with limited degradation