Biochemical characterization of important proteolytic enzymes from Aspergillus oryzae revealed by comparative...

This study employed an integrated approach of comparative transcriptomics and heterologous expression to identify and characterize important proteolytic enzymes from Aspergillus oryzae for enhancing doubanjiang fermentation. Comparative transcriptomic analysis between A. oryzae BL18 and 3.042 during koji-making identified 12 differentially expressed proteolytic enzyme genes. These candidate genes were heterologously expressed in Komagataella phaffii GS115 for enzymatic property characterization. Two proteolytic enzymes with relatively good stability under high salinity were identified. GME1468_g and GME389_g enzymes could retain 54.52 % and 28.42 % of their activities after treatment in 18 % salinity for 7 days, respectively. Furthermore, GME389_g enzyme was beneficial for the hydrolysis of globulin while GME1468_g enzyme preferred albumin hydrolysis. Based on these characterized properties, GME1468_g and GME389_g enzymes were applied individually and in combination in doubanjiang fermentation. The application of GME1468_g enzyme yielded the most significant improvement in product quality, resulting in the highest concentrations of amino acid nitrogen (0.88 g/100 g) and free amino acids (23.27 g/100 g), which were 66.04 % and 30.21 % higher than those in control group (0.53 g/100 g and 17.87 g/100 g, p < 0.05). This group also exhibited a significantly increased concentration of total volatile flavor compounds while nine key volatile flavor compounds in doubanjiang. The addition of GME1468_g enzyme also led to better sensory characteristics with stronger jiang aroma with clearly perceptible floral and fruity aroma. This work established a methodological framework for mining functional enzymes from key microbes and demonstrated its successful application in traditional food fermentation.