Enzymatic kinetics of β-conglycinin using alkaline protease from Bacillus subtilis ACCC 01746 and analysis of antigenicity of hydrolyzed peptide

Abstract

β-Conglycinin, the main protein of soybean, is a key allergen that causes soybean allergies, and hydrolysis is usually applied to lower its antigenicity. We evaluated the enzymolysis characters of β-conglycinin from the perspective of enzymolysis kinetics using alkaline protease from B. subtilis ACCC 01746. A dynamic model describing the hydrolysis of β-conglycinin was proposed using the initial substrate concentration, enzyme dosage (enzyme to substrate ratio) and hydrolysis time as variables to illustrate the kinetic behavior of enzymatic hydrolysis. The hydrolysis of soybean β-conglycinin was carried out at 60 g/L protein concentration, 0.6% enzyme dosage, 55 °C and pH 8.5 to observe the peptides with anti-enzymatic activities. The hydrolysates were gradually fractionated by ultrafiltration through cut-off membranes with molecular weights of 40, 30, 20, and 10 kDa, and their antigenicities were evaluated using indirect competitive enzyme-linked immunosorbent assay. The results showed that the degree of hydrolysis (DH) of β-conglycinin decreased as the β-conglycinin concentration (S₀) increased, but increased with enzyme dosage (E₀) increasing. Thus, the enzymatic hydrolysis of β-conglycinin followed the first-order kinetics model. The hydrolysis rate (V) was (527.89C_E0–2.5533C_S0) exp (–0.022DH), the DH-hydrolysis time was 45.454ln[1 + (11.614C_E0/C_S0–0.0562)t], and the correlated kinetic constants k₂ and k_d were 527.89 min⁻¹ and 8.6126 min⁻¹, respectively. The hydrolysis behavior of β-conglycinin varied considerably among the α', α, and β subunits. Faster hydrolysis rates were observed for the α' and α subunits compared to the β subunit. The relative molecular weights of the intercepted peptides from the hydrolysates were 14.8–40.1 kDa, and the antigenicity of the peptides with smaller molecular weight was reduced, but not removed completely. However, the alkaline protease from the strain appeared to effectively reduce the allergenicity of β-conglycinin. Therefore, it is possible to produce less allergenic soybean proteins using enzymatic hydrolysis. Additionally, the microbial alkaline protease may serve as a potential novel food enzyme and should be evaluated for the development of hypoallergenic foods.