Efficient expression and characterization of an acid protease in Aspergillus niger for bread making and soy protein modification

Acid proteases are important enzymes in the food industry. In this study, an acid protease (AopepA) from Aspergillus oryzae was high-level expressed extracellularly in Aspergillus niger using different expression strategies. The highest protease activity of 407.6 U/mL was yielded in a shake flask by the engineered A. niger strain, which was constructed using a mixed expression strategy of constitutive and induction types. High protease activity of 1153.6 U/mL with a protein concentration of 3.2 mg/mL was produced by fed-batch fermentation in a 5 L fermenter. AopepA displayed optimal activity at pH 2.5 and 50 ℃, respectively. It was stable within pH 2.0‒7.0 and up to 45 ℃. AopepA showed a broad substrate specificity and the highest specific activity of 920.8 U/mg towards hemoglobin. The addition of AopepA (1000 U/kg) during the bread making improved the specific volume by 16.6% and decreased the hardness by 26.9%. Moreover, AopepA was applied to modify soy protein, resulting in a 2.2-fold increase in foaming capacity and a 1.2-fold increase in foaming stability. The emulsifying activity and emulsion stability indexes were enhanced by 1.3- and 2.8-fold, respectively. This study provides a valuable way for the efficient expression of proteases in A. niger and broadens the application of acid proteases in the food industry.