Characterization and Industrial Application of the Enzyme Papain Through the Use of a Biosensor

Casein influences coagulation, yield, and product quality. This study aimed to develop and validate a papain (EC 3.4.22.2)-based biosensor to quantify casein in whole milk, evaluating its kinetic behavior and comparing it with HPLC. Papain was immobilized on nylon membranes and a cassava starch biopolymer. Enzyme kinetics (KM), stability, sensitivity, precision, and linearity of the system were evaluated. A t-test (α = 0.05) was applied to compare the casein concentration obtained by the biosensor versus HPLC (n = 20). Interference from milk proteins and calcium was also analyzed. The biosensor showed high affinity for casein (KM = 0.037 mM), with a linear range of 0.001-0.03 mM (R² = 0.9974) and a response time <5 s. No significant differences were found with HPLC (p = 0.0665). Stability reached 70 days at 4°C and was reusable up to 15 times. The developed biosensor proved to be an accurate, fast, and sustainable analytical alternative for the determination of casein in complex milk matrices.