The highest alkaline protease activity was 41.35 U/g dry weight at 72 h of fermentation in 60% soybean sample (Fig. 1d). Whereas both neutral and alkaline protease activities were lower in the 40% and 50% soybean sample. The previous study reported that production of enzymes required amino acids by digesting proteins in substrate. A. oryzae can utilize a pool of nitrogen sources in soybean to produce amino acids, it decreases the total protein content of soybean koji during cultivation [3, 5].