Abstract

• This study focuses on the isolation, purification, and characterization of alkaline protease enzyme (EC 3.4.21.63) from soybean. The enzyme was extracted using a Tris base buffer solution and purified through 30–60% ammonium sulfate precipitation followed by gel filtration chromatography on a Sephadex G-100 column. The purification process achieved a 3-fold increase in purity with a protein recovery rate of 5.8%. Qualitative confirmation of the enzyme was demonstrated using a ninhydrin test, while the enzyme activity was quantified at 2734.96 EU/g. Characterization of the enzyme revealed an optimum pH of 9.0 and an optimum temperature of 55 °C. Enzyme kinetics were studied, yielding an activation energy (Ea) of 4.553 kcal/mol, a Michaelis constant (Km) of 0.67 × 10⁻² g/mL, and a maximum velocity (Vmax) of 60.97 × 10⁻³ mM/min using Lineweaver-Burk plot. The reaction order was determined to be first-order. The enzyme exhibited pH stability and thermostability under tested conditions. Finally, the potential application of the crude alkaline protease enzyme in the detergent industry was evaluated, showing its effectiveness as a laundry detergent additive. These findings highlight its practical utility in industrial applications, particularly in environmentally friendly cleaning formulations.