The purpose of this study was to isolate and identify a strain with high production of streptokinase, and to analyze some characteristics of the streptokinase produced, including type, relative molecular mass, and fermentation curve. A high plasmin-production strain MX-6 was separated from Chinese douchi, which was identified as Bacillus subtilis by individual morphology, colony morphology, and homology comparison of 16S rDNA. The gene encoding plasmin in B. subtilis MX-6 (1 473 bp) was cloned by polymerase chain reaction (PCR). According to the results of multiple sequence alignment and phylogenetic tree between the deduced amino acid sequence and homologous sequences, the plasmin was identified as a nattokinase. The relative molecular mass of nattokinase produced by B. subtilis MX-6 was approximately 28 ku by SDS-polyacrylamide gels electrophoresis (SDS-PAGE), which coincided with the predicted molecular weight of 27 712.72 u. The fermentation curve of the nattokinase produced demonstrated that its maximum synthetic amount was obtained at 72 h, and the diameter of clear zone on the plasminogen-free fibrin plate was 21.60 mm. The results provide some theoretical basis and method reference for the development and application of nattokinase.
MAN Lili
,
XIANG Dianjun
. Partial characteristics of nattokinase produced by Bacillus subtilis MX-6[J]. Food and Fermentation Industries, 2019
, 45(6)
: 42
-47
.
DOI: 10.13995/j.cnki.11-1802/ts.019194
[1] WANG H, SUN X, WANG L, et al. Coproduction of menaquinone-7 and nattokinase by Bacillus subtilis using soybean curd residue as a renewable substrate combined with a dissolved oxygen control strategy[J]. Annals of Microbiology, 2018, 68(10): 655-665.
[2] FURIE B, FURIE B C. Mechanisms of thrombus formation[J]. New England Journal of Medicine, 2008, 359(9): 938-949.
[3] MOHANASRINIVASAN V, MOHANAPRIYA A, POTDAR S, et al. In vitro and in silico studies on fibrinolytic activity of nattokinase: A clot buster from Bacillus sp.[J]. Frontiers in Biology, 2017, 12(3): 219-225.
[4] CAI D, ZHU C, CHEN S. Microbial production of nattokinase: Current progress, challenge and prospect[J]. World Journal of Microbiology and Biotechnology, 2017, 33(5): 84.
[5] VAITHILINGAM M, CHANDRASEKARAN S D, GUPTA S, et al. Extraction of nattokinase enzyme from Bacillus cereus isolated from rust[J]. National Academy Science Letters, 2016, 39(4): 263-267.
[6] SUBATHRA D C, MOHANASRINIVASAN V, SHARMA P, et al. Production, purification and stability studies on nattokinase: A therapeutic protein extracted from mutant Pseudomonas aeruginosa CMSS isolated from bovine milk[J]. International Journal of Peptide Research and Therapeutics, 2016, 22(2): 263-269.
[7] DABBAGH F, NEGAHDARIPOUR M, BERENJIAN A, et al. Nattokinase: Production and application[J]. Applied Microbiology and Biotechnology, 2014, 98(22): 9 199-9 206.
[8] WENG M, DENG X, WU J, et al. Thermostability of subtilisin nattokinase obtained by site-directed mutagenesis[J]. Wuhan University Journal of Natural Sciences, 2014, 19(3): 229-234.
[9] ZHANG F, ZHANG J, LINHARDT R J. Interactions between nattokinase and heparin/GAGs[J]. Glycoconjugate Journal, 2015, 32(9): 695-702.
[10] 王家政,范明. 蛋白质技术手册[M]. 北京:科学出版社, 2001:77-100.
[11] WEI X, LUO M, XIE Y, et al. Strain screening, fermentation, separation, and encapsulation for production of nattokinase functional food[J]. Applied Biochemistry and Biotechnology, 2012, 168(7): 1 753-1 764.
[12] THOKCHOM S, JOSHI S R. Screening of fibrinolytic enzymes from lactic acid bacterial isolates associated with traditional fermented soybean foods[J]. Food Science and Biotechnology, 2014, 23(5): 1 601-1 604.
[13] LU F, SUN L, LU Z, et al. Isolation and identification of an endophytic strain EJS-3 producing novel fibrinolytic enzymes[J]. Current Microbiology, 2007, 54(6): 435-439.
[14] KOTB E. Purification and partial characterization of a chymotrypsin-like serine fibrinolytic enzyme from Bacillus amyloliquefaciens FCF-11 using corn husk as a novel substrate[J]. World Journal of Microbiology & Biotechnology, 2014, 30(7): 2 071-2 080.
[15] 李建强,石洪凌,肖冬光. 一种纳豆芽孢杆菌的分子鉴定及纳豆激酶基因克隆[J]. 唐山师范学院学报, 2013, 35(2): 37-40.
