Abstract
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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Afifah DN, Sulchan M, Syah D, Yanti SMT, Kim JH (2014) Purification and characterization of a fibrinolytic enzyme from Bacillus pumilus 2.g isolated from Gembus, and Indonesian fermented food. Prev Nutr Food Sci 19:213–219
Bradford MM (1976) Rapid and sensitive methods for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Chang CT, Fan MH, Kuo FC, Sung HY (2000) Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMRNK1. J Agric Food Chem 48:3210–3216
Chang CT, Wang PM, Hung YF, Chung YC (2012) Purification and biochemical properties of a fibrinolytic enzyme from Bacillus subtilis-fermented red bean. Food Chem 133:1611–1617
Chen H, McGowan EM, Ren N, Lal S, Nassif N, Shad-Kaneez F, Qu X, Lin Y (2018) Nattokinase: a promising alternative in prevention and treatment of cardiovascular diseases. Biomark Insights 13:1–8
Choi NS, Yoo KH, Yoon KS, Chang KT, Maeng PJ, Kim SH (2005) Identification of recombinant subtilisins. J Microbiol Biotechnol 15:35–39
Dong Z, Chen X, Cai K, Chen Z, Wang H, Jin P, Liu X, Permaul K, Singh S, Wang Z (2018) Exploring the metabolomic responses of Bacillus licheniformis to temperature stress by gas chromatography/mass spectrometry. J Microbiol Biotechnol 28:473–481
Fujita M, Nomura K, Hong K, Ito Y, Asada A, Nishimuro S (1993) Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan. Biochem Biophys Res Commun 197:1340–1347
Fukui Y, Yoshida M, Shozen KI, Funatsu Y, Takeno T, Oikawa H, Yano Y, Satomi M (2012) Bacterial communities in fish sauce mash using culture-dependent and –independent methods. J Gen Appl Microbiol 58:273–281
Guan L, Cho KH, Lee JH (2011) Analysis of the cultivable bacterial community in jeotgal, a Korean salted and fermented seafood, and identification of its dominant bacteria. Food Microbiol 28:101–113
Hadjiaj R, Badis A, Mechri S, Eddouaouda K, Khelouia L, Annane R, Hattab ME, Jaouadi B (2018) Purification, biochemical, and molecular characterization of novel protease from Bacillus licheniformis strain K7A. Int J Biol Macromol 114:1033–1048
Hsu TC, Yi PJ, Lee TY, Liu JR (2018) Probiotic characteristics and zearalenone removal ability of a Bacillus licheniformis strain. PLoS One. https://doi.org/10.1371/journal.pone.0194866
Hua Y, Jiang B, Mine Y, Mu W (2008) Purification and characterization of a novel fibrinolytic enzyme from Bacillus sp. nov. SK006 isolated from an Asian traditional fermented shrimp paste. J Agric Food Chem 56:1451–1457
Jeong SJ, Kwon GH, Chun JY, Kim JS, Park CS, Kwon DY, Kim JH (2007) Cloning of fibrinolytic enzyme gene from Bacillus subtilis isolated from cheonggukjang and its expression in protease-deficient Bacillus subtilis strains. J Microbiol Biotechnol 17:1018–1023
Jeong SJ, Cho KM, Lee CK, Kim GM, Shin JH, Kim JS, Kim JH (2014) Overexpression of aprE2, a fibrinolytic enzyme gene from Bacillus subtilis CH3-5, in Escherichia coli and the properties of AprE2. J Microbiol Biotechnol 24:969–978
Jo HD, Kwon GH, Park JY, Cha J, Song YS, Kim JH (2011) Cloning and overexpression of aprE3-17 encoding the major fibrinolytic protease of Bacillus licheniformis CH3-17. Biotechnol Bioprocess Eng 18:352–359
Kim GM, Lee AR, Lee KW, Park JY, Chun J, Cha J, Song YS, Kim JH (2009) Characterization of a 27 kDa fibrinolytic enzyme from Bacillus amyloliquefaciens CH51 isolated from cheonggukjang. J Microbiol Biotechnol 19:997–1004
Kim WK, Choi KH, Kim YT, Park HH, Choi JY, Lee YS, Oh HI, Kwon IB, Lee SY (1996) Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK11-4 screened from chungkook-jang. Appl Environ Microbiol 62:2482–2488
Koo OK, Lee SJ, Chung KR, Jang DJ, Yang HJ, Kwon DY (2016) Korean traditional fermented fish products: jeotgal. J Ethn Foods 3:107–116
Kwon GH, Lee HA, Park JY, Kim JS, Lim J, Park CS, Kwon DY, Kim YS, Kim JH (2009) Development of a RAPD-PCR method for identification of Bacillus species isolated from cheonggukjang. Int J Food Microbiol 129:282–287
Lan R, Kim IH (2018) Effects of Bacillus licheniformis and Bacillus subtilis complex on growth performance and faecal noxious gas emissions in growing-finishing pigs. J Sci Food Agric 99:1554–1560
Lee J, Xiang L, Byambabaatar S, Kim H, Jin KS, Ree M (2019a) Bacillus licheniformis α-amylase: structural feature in a biomimetic solution and structural changes in extrinsic conditions. Int J Biol Macromol 127:286–296
Lee NK, Kim WS, Paik HD (2019b) Bacillus strains as human probiotics: characterization, safety, microbiome, and probiotic carrier. Food Sci Biotechnol 28:1297–1305
Li Y, Liu M, Zhou J, Hou B, Su X, Liu Z, Yuan J, Li M (2019) Bacillus licheniformis Zhengchangsheng® attenuates DSS-induced colitis and modulates the gut microbiota in mice. Benef Microbes 10:543–553
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2- △△CT method. Methods 25:402–408
Meng L, Feldman L (2010) A rapid TRIzol-based two-step method for DNA-free RNA extraction from Arabidopsis siliques and dry seeds. Biotechnol J 5:183–186
Park MR, Oh S, Son SJ, Park DJ, Oh S, Kim SH, Jeong DY, Oh NS, Lee Y, Song M, Kim Y (2015) Bacillus licheniformis isolated from traditional Korean food resources enhances the longevity of Caenorhabditis elegans through serotonin signaling. J Agric Food Chem 63:10227–10233
Peng Y, Huang Q, Zhang R, Zhang YZ (2003) Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douchi, a traditional Chinese soybean food. Comp Biochem Physiol Biochem Mol Biol 134:45–52
Schallmey M, Singh A, Ward OP (2004) Developments in the use of Bacillus species for industrial production. Can J Microbiol 50:1–17
Shaw A, Bott R, Day AG (1999) Protein engineering of alpha-amylase for low pH performance. Curr Opin Biotechnol 10:349–352
Song CW, Rathnasingh C, Park JM, Lee J, Song H (2018) Isolation and evaluation of Bacillus strains for industrial production of 2,3-butanediol. J Microbiol Biotechnol 28:409–417
Wang C, Du M, Zheng D, Kong F, Zu G, Feng Y (2009) Purification and characterization of nattokinase from Bacillus subtilis Natto B-12. J Agric Food Chem 57:9722–9729
Wang CT, Ji BP, Li B, Nout R, Li PL, Ji H, Chen LF (2006) Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional douchi. J Ind Microbiol Biotechnol 33:750–758
Weng Y, Yao J, Sparks S, Wang KY (2017) Nattokinase: an oral antithrombotic agent for the prevention of cardiovascular disease. Int J Mol Sci 18:E523
Yao Z, Liu X, Shim JM, Lee KW, Kim HJ, Kim JH (2017) Properties of a fibrinolytic enzyme secreted by Bacillus amyloliquefaciens RSB34, isolated from Doenjang. J Microbiol Biotechnol 27:9–18
Zhu S, Cai D, Liu Z, Zhang B, Li J, Chen S, Ma X (2018) Enhancement of bacitracin production by NADPH generation via overexpressing glucose-6-phosphate dehydrogenase Zwf in Bacillus licheniformis. Appl Biochem Biotechnol 187:1502–1514
Funding
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.
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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 66, 787–795 (2021). https://doi.org/10.1007/s12223-021-00878-w
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DOI: https://doi.org/10.1007/s12223-021-00878-w