2019 ; Vol.29-3: 347~356
|Author||Zhuang Yao, Jeong A Kim, Jeong Hwan Kim|
|Place of duty||Gyeongsang National University, Jinju 52828, Korea, |
|Title||Characterization of a Fibrinolytic Enzyme Secreted by Bacillus velezensis BS2 Isolated from Sea Squirt Jeotgal|
J. Microbiol. Biotechnol.2019 ;
|Abstract||Bacillus sp. BS2 showing strong fibrinolytic activity was isolated from sea squirt (munggae)
jeotgal, a traditional Korean fermented seafood. BS2 was identified as B. velezensis by
molecular biological methods. B. velezensis BS2 grows well at 15% NaCl and at 10oC. When
B. velezensis BS2 was cultivated in TSB broth for 96 h at 37oC, the culture showed the highest
fibrinolytic activity (131.15 mU/μl) at 96 h. Three bands of 27, 35 and 60 kDa were observed
from culture supernatant by SDS-PAGE, and fibrin zymography showed that the major
fibrinolytic protein was the 27 kDa band. The gene (aprEBS2) encoding the major fibrinolytic
protein was cloned, and overexpressed in heterologous hosts, B. subtilis WB600 and E. coli
BL21 (DE3). B. subtilis transformant showed 1.5-fold higher fibrinolytic activity than
B. velezensis BS2. Overproduced AprEBS2 in E. coli was purified by affinity chromatography.
The optimum pH and temperature were pH 8.0 and 37oC, respectively. Km and Vmax were
0.15 mM and 39.68 μM/l/min, respectively, when N-succinyl-Ala-Ala-Pro-Phe-pNA was used
as the substrate. AprEBS2 has strong α-fibrinogenase and moderate β-fibrinogenase activity.
Considering its high fibrinolytic activity, significant salt tolerance, and ability to grow at 10oC,
B. velezensis BS2 can be used as a starter for jeotgal.|
|Key_word||Bacillus velezensis, fibrinolytic activity, sea squirt jeotgal, salt tolerance|
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.
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.
Fukui Y, Yoshida M , Shozen KI, Funatsu Y , Takano T, Oikawa H, et al. 2012. Bacterial communities in fish sauce mash using culture-dependent and –independent methods. J. Gen. Microbiol. 58: 273-281.
Han KI, Kim YH, Hwang SG, Jung EG, Patnaik BB, Han YS, et al. 2014. Bacterial community dynamics of salted and fermented shrimp based on denaturing gradient gel electrophoresis. J. Food Sci. 79: M2516-M2522.
Lee SH, Jung JY, Jeon CO. 2015. Bacterial community dynamics and metabolite changes in myeolchi-aekjeot, a Korean traditional fermented fish sauce, during fermentation. Int. J. Food Microbiol. 203: 15-22.
Lee Y, Cho Y, Kim E , Kim HJ, Kim HY. 2018. Identification of lactic acid bacteria in galchi- and myeolchi-jeotgal by 16S rRNA gene sequencing, MALDI-TOF mass spectrometry, and PCR-DGGE. J. Microbiol. Biotechnol. 28: 1112-1121.
Yongsawatdigul J, Rodtong S. Raksakulthai N. 2007. Acceleration of Thai fish sauce fermentation using proteinases and bacterial starter cultures. J. Food Sci. 72: 382-390.
Udomsil N, Rodtong S, Choi YJ, Hua Y, Yongsawatdigul J. 2011. Use of Tetragenococcus halophilus as a starter culture for flavor improvement in fish sauce fermentation. J. Agric. Food Chem. 59: 8401-8408.
Yoshikawa S, Kurihara H, Kawai Y, Yamazaki K, Tanaka A, Nishikiori T, Ohta T. 2010. Effect of halotolerant starter microorganisms on chemical characteristics of fermented chum salmon (Oncorhynchus keta) sauce. J. Agric. Food Chem.58: 6410-6417.
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: 523
Cai D , Zhu C , Chen S. 2 017. M icrobial p roduction of nattokinase: current progress, challenge and prospect. World J. Microbiol. Biotechnol. 33: 84.
Mine Y, Wong AHK, Jiang B. 2005. Fibrinolytic enzymes in Asian traditional fermented foods. Food Res. Int. 38: 243-250.
Kim J A, Yao Z , Kim HJ, Kim J H. 2 018. P roperties of g ul jeotgal (oyster jeotgal) prepared with different types of salt and Bacillus subtilis JS2 as starter. Microbiol. Biotechnol. Lett.46: 1-8.
Yao Z , Liu X, Shim JM, Lee KW, Kim H J, Kim JH. 2017. Properties of a fibrinolytic enzyme secreted by Bacillus amyloliquefaciens RSB34, isolated from Doenjang. J. Microbiol. Biotechnol. 27: 9-18.
Kwon GH, Lee HA, Park JY, Kim JS, Lim JK, Park CS, et al. 2009. Development of a RAPD-PCR method for identification of Bacillus species isolated from cheonggukjang. Int. J. Food Microbiol. 129: 282-287.
Kim GM, Lee AR, Lee KW, Park JY, Chun J, Cha J , et al. 2009. Characterization of a 27 kDa fibrinolytic enzyme from Bacillus amyloliquefaciens CH51 isolated from cheonggukjang. J. Microbiol. Biotechnol. 19: 997-1004.
Dower WJ, Miller JF, Ragsdale CW. 1988. High efficiency transformation of E. coli by high voltage electroporation. Nuclelic Acids Res. 16: 6127-6145.
Sambrook J, Russel DW. 2001. Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
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.
Fan B, Blom J, Klenk HP, Borriss R. 2017. Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus siamensis form an operational group B. amyloliquefaciens” within the B. subtilis species complex. Front. Microbiol. Doi. 10.3389/fmicb.2017.00022
Dunlap CA, Kim S J, Kwon SW, Rooney AP. 2 016. Bacillus velezensis is not a later heterotypic synonym of Bacillus amyloliquefaciens; Bacillus methylotrophicus, Bacillus amyloliquefaciens subsp. plantarum and ‘Bacillus oryzicola’ are later heterotypic synonyms of Bacillus velezensis based on phylogenomics. Int. J. Syst. Evol. Microbiol. 66: 1212-1217.
Palazzini JM, Dunlap CA, Bowman MJ, Chulze SN. 2016. Bacillus velezensis RC218 as a biocontrol agent to reduce Fusarium head blight and deoxynivalenol accumulation:genome sequencing and secondary metabolite cluster profiles. Microbiol. Res. 191: 30-36.
Chen L, Heng J, Qin S, Bian K. 2018. A comprehensive understanding of the biocontrol potential of Bacillus velezensis LM2303 against Fusarium head blight. PLoS One 13: e0198560. doi: 10.1371/journal.pone.0198560.
Kim B K, Kim H J, Lee JW. 2 013. Rapid s tatistical optimization of cultural conditions for mass production of carboxymethylcellulase by a newly isolated marine bacterium, Bacillus velezensis A-68 from rice hulls. J. Life Sci. 23: 757-769.
Liu X, Ren B, Chen M, Wang H, Kokare CR, Zhou X, et al. 2010. Production and characterization of a group of bioemulsifiers from the marine Bacillus velezensis strain H3. Appl. Microbiol. Biotechnol. 87: 1881-1893.
Yao Z, Kim JA, Kim JH. 2018. Gene cloning, expression, and properties of a fibrinolytic enzyme secreted by Bacillus pumilus BS15 isolated from gul (oyster) jeotgal. Biotechnol. Bioprocess Eng. 23: 293-301.
Heo K , Cho KM, Lee CK, Kim GM, Shin JH, Kim JS, et al. 2013. Characterization of a fibrinolytic enzyme secreted by Bacillus amyloliquefaciens CB1 and its gene cloning. J. Microbiol. Biotechnol 23: 974-983.
Jo HD, Lee HA, Jeong SJ, Kim JH. 2011. Purification and characterization of a major fibrinolytic enzyme from Bacillus amyloliquefaciens MJ5-41 isolated from meju. J. Microbiol. Biotechnol. 21: 1166-1173.
Peng Y, Yang X, Zhang Y. 2005. Microbial fibrinolytic enzymes: an overview of source, production, properties, and thrombolytic activity in vivo. Appl. Microbiol. Biotechnol. 69: 126-132.
Wu XC, Lee W, Tran L, Wong SL. 1991. Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases. J. Bacteriol. 173: 4952-4958.
Jeong SJ, Heo K, Park JY, Lee KW, Park JY, Joo SH, et al. 2015. Characterization of AprE176, a fibrinolytic enzyme from Bacillus subtilis HK176. J. Microbiol. Biotechnol. 25: 89-97.
Kim W, Choi K, Kim Y, Park H, Choi J, Lee Y, et al. 1996. Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang. Appl. Environ. Microbiol. 62: 2482-2488.
Wang CT, Ji BP, Li B, Nout R, Li PL, Ji H, et al. 2006. Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi. J. Ind. Microbiol. Biotechnol. 33: 750-758.