Abstract: In this study, 65 strains of dominant bacteria were isolated from Sojae Semen Praeparatum (SSP) from different regions and screened for their fibrinolytic enzyme activity by enzyme-linked immunosorbent assay (ELISA). Bacillus velezensis 20-1 was selected from these strains for its high fibrinolytic enzyme activity, and its fibrinolytic enzyme gene was amplified and heterologously expressed in Pichia pastori. As a result, a recombinant strain with high fibrinolytic enzyme activity, P. pastoris pPIC9K-his-aprEBS2, was obtained. The results showed that the gene expression level of P. pastoris pPIC9K-his-aprEBS2 was 5.8 times as high as that of B. velezensis 20-1, and the enzyme activity reached (3 025.59 ± 201.27) U/mL after 48 h culture, which was 8.9 times as high as that of B. velezensis 20-1. The fibrinolytic enzyme activity increased to (8 698.24 ± 180.73) U/mL under optimized conditions. The acute toxicity test showed that the recombinant bacterium was non-toxic. SSP fermented by the recombinant bacterium was (17 287.02 ± 103.20) U/g. Physicochemical characterization, sensory evaluation and electronic tongue analysis demonstrated that the recombinant bacterium could improve the sensory quality and flavor of SSP. The thrombolysis rate of SSP fermented by it was (60.55 ± 1.58)%, which was higher than that of commercially available SSP.

Key words: Douchi fibrinolytic enzyme; heterologous expression; Pichia pastoris; Sojae Semen Praeparatum