Bacillus licheniformis HJ4 showing strong fibrinolytic activity was isolated from Hwangseokae jeotgal. aprEHJ4, a major fibrinolytic gene, was cloned by PCR, and an ORF consisting of 379 amino acids was located. The mature enzyme was expected to be 27 kDa in size after processing, but a 24-kDa protein was observed by SDS-PAGE and fibrin zymography, indicating additional processing. RT-qPCR showed that expression level of aprEHJ4 in culture with 0% salt (control) was the highest followed by culture with 8% salt (89.7% of control) and 5% salt (74.2%) at 84 h. The expression level in culture with 15% salt was 46.9%. The results matched with the fibrinolytic activity measurements of cultures and indicated that AprEHJ4 maintained significant activity in the presence of salt up to 15% (w/v). AprEHJ4 was overproduced in Escherichia coli, and mature 27 kDa protein was purified after in vitro renaturation. The optimum pH and temperature of AprEHJ4 were pH 8 and 40 ℃, respectively.
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This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2017R1D1A1B03030037). Meng Y, Yao Z, Lee SJ, and Yoo JY were supported by BK21 program, MOE, Republic of Korea. Le HG and Jeon HS were supported by full-time graduate student scholarship from Gyeongsang National University.
The authors declare no competing interests.
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Meng, Y., Yao, Z., Le, H.G. et al. Characterization of a salt-resistant fibrinolytic protease of Bacillus licheniformis HJ4 isolated from Hwangseokae jeotgal, a traditional Korean fermented seafood. Folia Microbiol (2021). https://doi.org/10.1007/s12223-021-00878-w