Proteolytic enzymes are important to modify the processing characteristic of soybeans and have been utilized for the production of soybean-derived foods-e.g., miso and soy sauce. Proteolytic enzymes produced in the cotyledons of soybean seeds during germination are used to modify the characteristics of processed foods. The optimum conditions for protease production during germination were determined and the characteristics of these proteases were described. The soybean variety Ohtsuru produced high levels of a semi-alkaline proteolytic enzyme under conditions of 20% CO2 and 25℃. The protease was purified using cation-exchange and size-exclusion chromatography. The monomeric protein had a mass of 84.9 kDa. The optimum pH and temperature for this protein were pH8.0 and 30℃, respectively, and the protein was stable at pH7.0-10.0 and 4-30℃. The proteolytic enzyme was activated by magnesium, zinc, and manganese ions and was inhibited by copper and mercury ions, EDTA, and EGTA. However, the enzyme was not inhibited by trypsin inhibitor. The enzyme cleaved substrates by endo-type proteolysis and cleaved the α-, α’-, and β-subunits of the soy protein β-conglycinin. The enzyme was able to modify the characteristics of soybean during their production. Moreover, it digested a major allergen protein, Gly m Bd60K, which is an α-subunit of beta-conglycinin.