Yao, Zhuang
(Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University)
,
Meng, Yu
(Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University)
,
Le, Huong Giang
(Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University)
,
Lee, Se Jin
(Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University)
,
Jeon, Hye Sung
(Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University)
,
Yoo, Ji Yeon
(Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University)
,
Afifah, Diana Nur
(Nutrition Science Department, Faculty of Medicine, Diponegoro University)
,
Kim, Jeong Hwan
(Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University)
Two Bacillus strains, K3 and K208, both demonstrating strong fibrinolytic activities were isolated from Kimchi, a traditional Korean preparation of fermented vegetables. Isolates were subjected to various molecular biology based identification methods including RAPD-PCR and identified as B. subtilis...
Two Bacillus strains, K3 and K208, both demonstrating strong fibrinolytic activities were isolated from Kimchi, a traditional Korean preparation of fermented vegetables. Isolates were subjected to various molecular biology based identification methods including RAPD-PCR and identified as B. subtilis and B. velezensis, respectively. Tryptic soy broth (TSB) was found to best maintain both the growth and the fibrinolytic activity of these strains. Culture supernatants were analyzed by SDS-PAGE and fibrin zymography, and the results indicate that a 40 and 27 kDa band seem to be responsible for the fibrinolytic activities of these two isolates and the 27 kDa band was subsequently identified as the mature form of AprE, the major fibrinolytic enzyme. Thus the aprE genes were cloned and the translated amino acid sequences demonstrated 99.3% identity with each other, and 86.5% identity with BsfA, a fibrinolytic enzyme from B. subtilis ZA400 also isolated from Kimchi, and AprE2, a fibrinolytic enzyme from B. subtilis CH3-5 isolated from Cheonggukjang, a traditional Korean fermented soy. Given this B. subtilis K3 and B. velezensis K208 may be promising starter cultures in the production of fermented foods.
Two Bacillus strains, K3 and K208, both demonstrating strong fibrinolytic activities were isolated from Kimchi, a traditional Korean preparation of fermented vegetables. Isolates were subjected to various molecular biology based identification methods including RAPD-PCR and identified as B. subtilis and B. velezensis, respectively. Tryptic soy broth (TSB) was found to best maintain both the growth and the fibrinolytic activity of these strains. Culture supernatants were analyzed by SDS-PAGE and fibrin zymography, and the results indicate that a 40 and 27 kDa band seem to be responsible for the fibrinolytic activities of these two isolates and the 27 kDa band was subsequently identified as the mature form of AprE, the major fibrinolytic enzyme. Thus the aprE genes were cloned and the translated amino acid sequences demonstrated 99.3% identity with each other, and 86.5% identity with BsfA, a fibrinolytic enzyme from B. subtilis ZA400 also isolated from Kimchi, and AprE2, a fibrinolytic enzyme from B. subtilis CH3-5 isolated from Cheonggukjang, a traditional Korean fermented soy. Given this B. subtilis K3 and B. velezensis K208 may be promising starter cultures in the production of fermented foods.
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성능/효과
Due to the fundamental role of RecA, recA gene is ubiquitous, and has been used as a phylogenetic marker for distantly related species [14]. The results showed that both 16S rRNA and recA gene sequences are highly conserved among these closely related Bacillus species, and the sequencing data alone was not enough for the accurate identification of bacilli isolates at species level [15]. RAPD-PCR was done to determine the species of K3 and K208.
후속연구
subtilis strains produce similar fibrinolytic enzymes, but still significant differences are observed among them. Therefore further studies are necessary to find out different enzymes in the primary structure have different roles during growth. The highly identical amino acid sequences indicates the highly conserved nature of AprEs among Bacillus species, implying the important roles of AprE for Bacillus species during stationary growth phase.
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