Two-Way Purification of Alkaline Phosphatase from Phaseolus vulgaris (Cranberry Group) Seeds: Sequential Low-Temperature Ultracentrifugation and Anion-Exchange FPLC with Biochemical Characterization and Kinetic Analysis

Alkaline phosphatase (3.1.3.1) (ALP) an enzyme central to phosphate metabolism, is known for its roles in bone mineralization and nutrient mobilization in addition to that it has crucial application in biotechnology and diagnostics. In this study, ALP was extracted from seeds of Phaselous Vulgaris (cranberry group). The crude protein extract (CPP) exhibited an activity of 9.51µmol/min/ml with specific activity 1.39 µmol/min/ml/mg. Two independent purification strategies were applied: (1) low temperature ultracentrifugation yielded molecular weight optimized concentrate (MWOCT) with 4.28 fold purification (specific activity 5.95 µmol/min/ml/mg, 128.3% activity recovery) and (2) anion-exchange FPLC (AKTA-Q) on 15 ml diluted crude (30 ml, Affinity chromatography load (AELXD load) produced a single major peak at retention 20.415 ml (area 117.2 mAU*ml, conductivity 22.89 mS/cm), yielding active fractions : Affinity chromatography eluate fraction 1 (AEET.1) and Affinity chromatography eluate fraction 2 (AEET.2) with retained activity (0.0275–0.0294 µmol/min/mg), indicating superior purity. Kinetic parameters were Vmax = 5.55 µmol/min and Km = 172.2 µM. Optimal pH was 10.0, temperature 40 ^o C, and substrate saturation at ~ 4.35 mM pNPP, SDS –PAGE showed progressive purification, with AEET.2 dominated by a single ~ 63–70 kDa band. Solution- based BCIP/NBT confirmation produced blue–purple coloration in all active fractions (strongest in MWOCT and AEET.2), verifying catalytic function retention. This parallel two-way approach achieved significant enrichment with high recovery and purity, offering a simple scalable strategy for plant enzyme isolation in resource limited settings.